Download Proteases and proteolytic cleavage of storage

Journal of Experimental Botany, Vol. 47, No. 298, pp. 605-622, May 1996
Journal of
Experimental
Botany
REVIEW ARTICLE
Proteases and proteolytic cleavage of storage proteins in
developing and germinating dicotyledonous seeds
K. Miintz1
Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466 Gatersleben, Germany
Received 25 July, 1995; Accepted 16 January 1996
Abstract
Proteolytic cleavage plays an important role in
storage protein deposition and reactivation in seeds.
Precursor polypeptides are processed by limited
proteolysis to mature subunits of reserve proteins in
storage tissue cells of developing seeds. Steps of proteolytic processing are closely related to steps in
intracellular protein transfer through the endomembrane system and to the deposition in the storage
vacuole. In germinating seeds special endopeptidases
trigger storage protein breakdown by limited proteolysis. The induced conformation changes of storage
proteins open them to attack by additional endo- and
exopeptidases which degrade the protein reserves
completely. Proteases that catalyse limited cleavage
or complete degradation are synthesized as precursors which also undergo stepwise limited proteolysis when they are formed in cotyledons of developing
or germinating seeds. In general, this processing
transforms enzymatically inactive proenzymes into
active proteases. Different compartments participate
in the processing steps. Many of the proteases are
encoded by small multigene families. Different members of the corresponding protease families seem
to act during seed development and germination.
Proteolytic processes that contribute to the molecular
maturation and to the reactivation of storage proteins
in dicotyledonous seeds seem to be controlled by (1)
differential expression of members of the proteaseencoding gene families; (2) stepwise processing and
activation of protease precursor polypeptides; (3) transient differential compartmentation of precursors and
mature polypeptides of proteases and storage proteins, respectively; and (4) interacting changes in
storage protein structure and protease action. The
present knowledge on these processes is reviewed.
' Fax: +49 03 94 82 5-366.
O Oxford University Press 1996
Key words: Dicotyledons, seeds, storage
proteolytic cleavage, proteases.
proteins,
Introduction
Developmental^ regulated changes in cellular protein
patterns result from the degradation of proteins which
are no longer needed, as well as from the biosynthesis of
new proteins. The latter depends on the supply of amino
acids which, at least in part, are recycled from protein
breakdown. Similarly, cellular protein patterns change in
response to environmental influences, like temperature
stress (heat shock proteins), water supply (desiccation
proteins), or pathogen attack (pathogenesis related (PR)
proteins). Developmentally and environmentally induced
changes in the cellular proteins take place at qualitative
(pattern changes) as well as quantitative levels, for
example, increase or decrease in enzyme activity by changing the amount of the corresponding protein. Protein
biosynthesis and degradation represent important factors
in the regulation of nitrogen sink/source relationships
which are controlled by developmental as well as environmental factors, such as the storage and reactivation of
vegetative storage proteins in soybean (Staswick, 1990,
1994) or storage proteins in seeds (Mflntz, 1994).
Misfolded and damaged proteins have to be continuously
eliminated by protein degradation and to be replaced by
newly formed proteins. In addition, limited proteolysis
contributes to precursor protein maturation and modification which often are related to intracellular protein
sorting and targeting processes, like the detachment of
signal peptides from precursor proteins that are
synthesized at membrane-bound polysomes and co-translationally sequestered into the lumen of the endoplasmic
reticulum (ER). Limited proteolysis and complete polypeptide degradation are closely interacting processes.
606 Muntz
Detached protein fragments like signal and additional
targeting peptides of secretory and vacuolar proteins, of
nuclear-encoded plastid and mitochondria! proteins or
the propeptides which result from the subsequent processing of various precursor polypeptides, for example,
from the activation of zymogens, undergo rapid breakdown. The major part of these protein precursors, however, remains functionally intact and might be further
transported into other cellular compartments.
Proteases, subclassified into endo- and exopeptidases,
catalyse limited and complete proteolysis (for reviews see
Barrett, 1994; Rawlings and Barrett, 1993). Different sets
of proteases and protein degradation systems have been
associated with different cellular compartments (Vierstra,
1993) like the ubiqutin-dependent protein breakdown
system and the proteasomes in the cytosol or the plastid
protein degradation system. A system should exist to
degrade glyoxysomal proteins when these microbodies
are transformed into peroxisomes in lipid storing cotyledons of germinating seeds. Vacuoles in storage tissues of
seeds, tubers or stems of trees harbour compartmentspecific sets of proteolytic enzymes that play a critical
role in processing and breakdown of storage protein
depositions (Mtlntz et al. 1985; Shutov and Vaintraub,
1987; Wilson, 1986).
Seed storage proteins in the restricted sense are globulins which are characteristic of dicotyledonous seeds and
a few cereals, like oats and rice, and the prolamins and
glutelins from cereal grains acccording to the classification
of Osborne (1924). They are only formed in specific
storage tissues (e.g. endosperm or cotyledon mesophyll)
during seed development. At the time of storage protein
accumulation the respective organs and storage tissues
represent nitrogen and carbon sinks. They become nitrogen and carbon sources when the protein reserves are
reactivated. Within the frame of changing sink/source
relations storage protein metabolism represents a special
example for the developmentally and environmentally
controlled formation and degradation of proteins. In the
storage tissue cells the storage vacuole harbours the
majority of participating compartment-specific proteolytic
enzymes.
Most storage proteins are multimeric. The subunits are
polymorphic and encoded by multigene families. The
primary translation products of storage protein mRNA
represent precursors of the subunits formed at membranebound polysomes (rough endoplasmic reticulum, rER).
Most of the precursor polypeptides (e.g. storage globulins)
are transported into the storage vacuole. On their way
they pass the Golgi apparatus, where at the trans-Golgi
they are entrapped into transfer vesicles which are then
targeted to the vacuole (for reviews see Chrispeels, 1991;
Chrispeels and Raikhel, 1992; Vitale et al, 1993). As
protein deposition proceeds the storage vacuole differentiates into protein bodies. In the endosperm of developing
maize kernels the protein bodies are directly formed from
the prolamin (zein)-accumulating ER (Larkins and
Hurkman, 1978). It has also been postulated that the
prolamin (gliadin) in wheat is directly transferred to
storage vacuoles without passing the Golgi apparatus
(Galili et al, 1995). Several of the globulin polypeptides
are modified by glycosylation in the ER and Golgi
apparatus and/or undergo additional processing by limited proteolysis in the vacuole. This latter step of molecular maturation then transforms the molecular species
which fits to intracellular transport into a species which
can be deposited in the vacuole. Clearly, the processing
of precursors of storage globulin subunits by limited
proteolysis is closely related to changing structure function relations which evolutionarily have been adapted on
the one side to the constraints acting during intracellular
protein transfer and targeting as well as on the other side
to the constraints governing deposition and accumulation
inside the storage vacuoles. This includes controlled limited proteolysis as well as the protection against premature
storage protein degradation which might be mediated by
special structural characters of the storage proteins,
by maintaining proteases in an inactive state and/or by
appropriate compartmentation.
A third group of constraints which have most certainly
played a role in the evolution of these proteins, as well
as the proteases that degrade them, are those imposed by
storage protein reactivation. The storage proteins must
be made susceptible to proteolytic attack by appropriate
structural changes, protease activation and changes in the
localization of various components among various compartmentations. Limited proteolysis is essential for the
initiation of storage protein breakdown. The triggering
endopeptidases may be present, in an inactive state with
their substrates inside the protein bodies or they may be
newly synthesized during seed germination. The structural
changes induced by the initial cleavages of the storage
proteins result in conformation changes that open them
to further degradation. In storage tissue cells this is
accompanied by the reformation of the vacuole from
protein bodies.
The processes of storage protein deposition and reactivation have been most extensively investigated using
globulins of dicotyledonous plants. Close analogues to
the globulins exist in the major storage proteins of oats
and rice, and in minor storage proteins of other monocotyledonous seeds. A knowledge of the structure of the
storage globulins is essential for understanding their
interactions with the proteases that degrade them.
Globulin structure
According to their sedimentation characteristics in sucrose
gradients dicotyledonous storage proteins are classified
into 1 IS, 7S and 2S proteins. Whereas 1 IS and 7S proteins
Proteolysis of seed storage proteins
belong to the globulins, except rice where the 1 IS protein
was classified as a glutelin, the 2S class comprises globulins as well as albumins. The 11S and 7S globulins are
named legumin-like or legumins and vicilin-like or vicilins,
respectively, in accordance to the predominating storage
globulins in pea (Pisum sativum L.) and faba beans (Vicia
faba L.). Trivial names, which frequently have been
derived from the botanical name of the corresponding
plant, are in use for many of these storage proteins.
Legumins (11S globulins)
Legumin holoproteins, which have a molecular weight of
300—400 kDa, are composed of six nearly identical subunits with molecular weights of 50-60 kDa. Each subunit
is composed of two differently sized polypeptide chains
(Fig. 1). The larger more hydrophilic one (mol. wt.
30-40 kDa) has a weak acidic pi and is named a-chain,
whereas the smaller more hydrophobic one (mol. wt.
20 kDa) has a strongly basic pi and is named /-chain.
Both chains are linked by a disulphide bridge between
cysteine residues at highly conserved positions in the a(amino acid residue 87) and /-chain (amino acid residue
7). The amino acid sequences of /-chains are more
homogeneous than those of the a-chains which vary
considerably in length because of different numbers of
repeats in its C-terminal part (Heim et al, 1989, 1994;
Horstmann et al, 1993). In addition, much larger achains are present in a minor group of large legumin
subunits which, for example, have been found in seeds of
Vicia faba L. The sequence of one of these large subunits
(mol. wt. 64 502 kDa) supports the hypothesis that the
larger subunits have evolved through amplification and
mutation of repeats in the C-terminal domain of the achain (Heim et ai, 1994). There is no indication that the
legumin holoprotein molecules represent homo-oligomers.
In the quaternary structure of the holoprotein, two trimers
are layered upon and twisted against each other at an
angle of 60°. The hydrophobic /-chains are mostly buried
inside the holoprotein. The more hydrophilic a-chain is
mainly located at the surface and forms loops which
extend out of the protein. The major loop is formed by
the variable and hydrophilic C-terminal region of the
a-chain.
Legumin genes and the corresponding mRNAs encode
subunits. Therefore, the primary translation product
corresponds to one subunit. It has a transient N-terminal
signal peptide. a- and /-chains are linked by a peptide
bond between the C-terminal amino acid residue of the
a-chain, which always is Asn, and the N-terminal residue
of the/-chain, which is almost always Gly. The disulphide
bridge between both the a- and /-chain regions of
the legumin precursor is already formed in the lumen of
the ER into which the growing pre-prolegumin is
inserted during biosynthesis. The signal peptide is
607
co-translationally detached and thereby prolegumin is
generated. Prolegumin assembles into trimers. These represent the transport-competent molecular form of legumin
precursors (Fig. 3). The peptide linkage between a- and
/-chains is only proteolytically cleaved after the prolegumin trimers have reached the storage vacuole. Since
both chains have already previously been bridged by the
disulphide linkage, a- and /-chains which are encoded by
one gene remain paired in the subunit. Prolegumins do
not seem to be able to assemble directly into hexamers.
Cleavage of the -Asn-Gly-linkage forms the prerequisite
for the transformation of two trimers into one hexamer.
Disassembly of the subunits does not seem to occur
during the trimer to hexamer transformation which results
from a very specific single proteolytic cleavage.
Vicilins (7S globulins) and their structural similarities to
legumins
Vicilin holoproteins which have molecular weights
between 150 and 240 kDa are trimers composed of two
types of subunits, large convicilin-like ones with mol. wt.
70-80 kDa and small subunits of about 50 kDa (Fig. 1).
Homo-oligomeric as well as hetero-oligomeric trimers are
known (Thanh and Shibasaki, 1977), the latter composed
of different ratios of large and small subunits (Mtintz
et al, 1986). Amino acid sequences of the large and small
subunits are very homologous. Close to their N-terminus
the large subunits contain an approximately 20 kDa large,
strongly hydrophilic insertion comprising repetitive elements. The major C-terminal 50 kDa fragment exhibits
high sequence similarity to the small subunits (Doyle
et al, 1986). The small subunits are polymorphic and
differ with respect to two characters: the degree of glycosylation and the presence of sites for limited proteolysis
by trypsin-like enzymes. Non-glycosylated subunits free
of cleavage sites have also been described, for example,
the major 50 kDa subunit from Vicia faba vicilin
(Bassuner et al., 1987; Weschke et al., 1988) and the
70 kDa convicilin subunit from peas, Pisum sativum L.
(Newbigin et al., 1990). Phaseolin, the best-known 7S
globulin from garden bean (Phaseolus vulgaris L.) is
composed of non-cleavable 50 kDa subunits with different
degrees of glycosylation (Slightom et al, 1985). There is
strong evidence that vicilin and legumin have a common
evolutionary origin (Shutov et al, 1995) and that their
subunits have principal similarity in the 3-dimensional
structure (Lawrence et al, 1994; Shutov et al, 1995)
which corresponds to the structure of phaseolin from the
garden bean (Lawrence et al, 1990, 1994) and canavalin
from jackbean, Canavalia ensiformis (L.) DC. (Ng
et al, 1993).
Like legumin subunits the vicilin subunits are encoded
by multigene families. Primary vicilin translation products
bear transient N-terminal signal sequences which are
608 Miintz
no.
processing scheme
subunit name
plant species
( processing enzyme )
1
N
a
p
C
I ,
subunits of tegunwv
the 12Sgfobulra
several lecteis
Pimm satnnjm, victa laba.
Glyctne max, Lupnus spec ,
Avena stbva. Oryza sattva
P&utn sMtrwm,
Rianus communs
(signal pepWase.
legumam)
2a
zeffi, hocdein
victtn
Pfl-congiyami
Zoa mays. Homeum yulpanj
Gtyctne max
(signal pepddase )
pnateoin
vtafin
2b
Phasa&us wtgam,
Paum saUvum, Victa laDa
(signal peptxjase)
Pisum sxtrvum, Viaa fatoa
2c
{signal popbdase,
unknown enzymes )
a ++p
p T
iL
3a
N
c
1
convicHln
Paum satiwm
Glyanemax
i
1
a- fl-congtyanm
o-p-cooglydtwi
i
1
a-gtobulln
1
(signal pepooase.
protsaseCI)
m
3b
1 1
1
N
1
N
|
m
c
1
(a
c
1
c
1
naptn,
2Sa«xjniin
Gossyptum twsutum
Brasses napus
Bertnofctto excelsa
N
c
1
NC
^1
\ pepteJaw.
legumatn,
unknown enzymes?)
1
N
c. 1
I
—i
/A\
C N "0
NC / \ r \ N C
concanavalmA
Canavaia enstfotmrs
(signal peptidase.
loguman )
Fig. I. Processing of different storage proteins by limited proteolysis during seed maturation. N and C indicate the N- and C-terminus of the
polypeptides: <j>. glycosylation site: the transient N-terminal signal peptide is similarly hatched in all cases, different hatchings were used for
propeptides. whereas the mature chains of the subunits remained white. I, 12S globulin processing; 2a-2c, different forms of the processing of
50 IcDa subunits of 7S globulins, 3a and 3b, different forms of the processing of 70 kDA subunits of 7S globulins, 4, 2S globulin processing; 5
concanavalin A processing.
Proteolysis of seed storage proteins
co-translationally detached when the growing polypeptide
is sequestered from the membrane-bound polysomes into
the ER-lumen. No disulphide bridges are known to link
7S globulin subunits. Core glycosylation of the respective
vicilin subunits takes place in the ER where trimers are
also assembled. Glycosyl side-chains are trimmed and
modified in the Golgi apparatus. Like prolegumin, vicilin
trimers are sorted into transfer vesicles at the trans-Golgi
network and transported into the storage vacuoles.
2S storage proteins
This is a heterogeneous group of proteins where polypeptides predominate which are structurally related to the
napin-like 2S proteins from Cruciferae (Shewry and
Tatham, 1990). 2S storage proteins are polymorphic and
encoded by multigene families. The proteins are monomeric and consist of disulphide-linked large and small
polypeptide chains (Fig. 1) which are derived from a
common precursor which bears a transient N-terminal
signal peptide. After its detachment the primary translation product undergoes an even more complicated proteolytic processing than prolegumin. In addition to the two
fragments which correspond to the mature large and
small polypetide chains, the precursor comprises a propeptide located between the signal peptide and the
N-terminus of the small polypeptide chain. The large
chain forms the C-terminal fragment in the precursor.
The propeptide cleavage site as well as the cleavage site
between the two polypeptide chains are flanked by Asnresidues in the P-position of many 2S protein precursors.
In the ER, the regions of the propolypeptide which
correspond to the small and large chains are already
linked by disulphide bridge formation. After the propeptide is cleaved off, the peptide linkage between the small
and large chain is cleaved. In the case of the 2S albumin
of Brazil nut (Bertholletia excelsa H.B.K.) this step
involves the elimination of a short linker peptide between
the small and large chains. Finally, the large chain of the
Brazil nut 2S albumin is trimmed by the detachment of
short oligopeptide fragments at the C-terminus.
609
peptide fragment are flanked by Asn-residues in the
P-position.
Processing of storage globulin precursors by
limited proteolysis in developing seeds
Signal peptide cleavage
Precursor polypeptides of storage globulins are sequestered from their cytoplasmic site of formation into the
ER-lumen, thus entering the so-called secretory pathway
through the endomembrane system. This membrane
transfer is mediated by the N-terminal signal sequence
which is proteolytically detached after finishing its function as a targeting and membrane transfer signal. The
single site cleavage is catalysed by a signal endopeptidase
located in the ER membrane and acting at its inner
surface (Dalbey and von Heijne, 1992). The enzyme has
been extensively investigated in eukaryotic animals and
yeast (Lively et al., 1994). It is a hetero-oligomeric serine
endopeptidase. The cleavage site between signal peptide
and N-terminus of the polypeptide chain is characterized
by a specific arrangement of amino acid residues described
by the -l;-3-rule according to von Heijne (1986). Since
signal peptides of plant proteins are correctly detached in
heterologous cell-free systems composed of the wheat
germ in vitro translation system, completed with dog
pancreas signal recognition particles and microsomal
membranes, as well as in homologous systems with plant
microsomes, a similar signal peptidase should also be
present in plants (Bassiiner et al., 1984). As a result of
the signal peptide cleavage a propolypeptide is formed in
most cases. As far as presently known, this propolypeptide
does not undergo further limited proteolysis in the ER
or Golgi compartment, although there the polypeptides
fold into a conformational state and oligomerize, which
makes them competent for intracellular transfer through
the endomembrane system and into the storage vacuole.
Several storage protein polypeptides are modified by
glycosylation during their passage through the ER and
Golgi compartment, but this glycosylation does not seem
to be a prerequisite for their intracellular transfer and
targeting (Voelker et al., 1989).
Concanavalin A
The sequence of mature concanavalin A (con A) is not
co-linear to the sequence of the precursor polypeptide
derived from the corresponding cDNA (Carrington et al.,
1984). After the N-terminal signal peptide is cotranslationally cleaved off in the rER, the glycosylated
procon A undergoes processing. An internal glycosylated
oligopeptide fragment is excised and the N- and
C-terminus of the precursor are ligated in a head-to-tail
position (Bowles et al, 1986; Faye and Chrispeels, 1987).
In addition, small terminal oligopeptide fragments might
be detached (Fig. 1). Both cleavage sites of the internal
Propolypeptide processing by limited proteolysis in
vacuoles
The presence of N-terminally Asn-flanked processing sites
is a common character of many US globulin (Lawrence
et al., 1994), 2S storage protein (Hara-Nishimura et al.,
19936) and even protein body membrane protein precursors (Inoue et al, 1995). The complete evolutionary
conservation of this Asn at the C-terminus of the large
legumin polypeptide chains indicates that it may be
important for recognition by the corresponding processing enzyme. Mutation or deletion of this Asn renders
610 Milntz
prolegumin uncleavable in vitro as well as in vivo.
Recently, cysteine endopeptidases have been described
from castor bean (Hara-Nishimura et al, 1991), soybean
(Scott et al, 1992; Muramatsu and Fukazawa, 1993) and
jack bean (Abe et al, 1993) that at least in vitro catalyse
the Asn-specific limited cleavage of prolegumins, precursors of 2S proteins, proconcanavalin A and some
other proteins. An enzyme belonging to this class of
endopeptidases, but prepared from germinating vetch
seeds, was able to catalyse in vitro the Asn-specific molecular maturation of prolegumin (Becker et al, 1995a), that
normally occurs in developing seeds. Polypeptides which
cross-react with antibodies raised against the enzyme
from castor bean were also detected in other plant organs
except seeds (Hiraiwa et al, 1993). Therefore, this type
of enzyme may belong to a class of cysteine endopeptidases which specifically cleaves at the C-terminus of Asn
residues that are exposed in a way which gives the
enzyme access.
Legumain in developing and mature seeds: When the
cDNA corresponding to the castor bean (Ricinus communis L.) Asn-specific processing proteinase from seeds
was sequenced and the complete amino acid sequence
was derived (Hara-Nishimura et al, 1993a, 1995), it
turned out to be a new type of cysteine endopeptidase.
The sequence did not correspond with the well-known
papain-like cysteine endopeptidases. The only similarity
that appeared on screening the databases was to the
sequence of an endopeptidase from Schistosoma mansoni,
a human parasite. The Ricinus mRNA encodes a 55 kDa
pre-propolypeptide that is presumably comprised of a 31
amino acid-long N-terminal signal peptide, the mature
37 kDa enzyme and an approximately 14 kDa C-terminal
propeptide sequence the exact length of which is unknown
since the C-terminus of the mature enzyme has yet to be
determined (Fig. 2b). Antibodies have been raised against
the purified native proteinase protein and used to localize
the enzyme to vacuoles in cells of the maturing castor
bean endosperm in which US and 2S storage proteins
are also localized. This result reinforces the interpretation
of previous 'grind and find' experiments in which the
Asn-specific processing enzyme co-purified with the protein bodies of castor bean endosperm. The same antibody
cross-reacted in immunoblots with polypeptide bands
after electrophoresis of extracts from mature and early
germinating seeds, too. Extracts from roots, hypocotyl
and leaves of castor bean as well as from pumpkin and
soybean cotyledons and hypocotyls, from mung bean
roots and spinach leaves were active in the enzyme-
PROTBNASE A
128
1 18
152
286
307
359
C C
QGQCGSCWAFST
TDLNHGVA
1
A
NSWG
/ \
KDEL
ER
i
?
?
VA?
PROTEINASE B
24
49
77
216
265
376 ( ? )
493
ND
NYRHQ.D.CHAY
EACESGS
TCLGDLYS
ER
Fig. 2. Schematic presentation of essential characters of proteinases A and B. SP, signal peptide; PP1 and PP2, propeptides; i£, glycosylation sites;
C, positions of cysteine residues; sequences of highly conserved fragments are indicated; arrows, amino acid residues of the active centre in papain
which are conserved in proteinase A; ER, endopiasmic reticujum, and VA, vacuole, indicate the cellular compartments where processing (presumably?)
occurs.
Proteolysis of seed storage proteins
specific assay which uses a synthetic decapeptide substrate
containing an Asn-flanked internal cleavage site (Hiraiwa
et al, 1993). The cDNA-derived amino acid sequence of
the vacuolar Asn-specific processing enzyme of soybean
cotyledons exhibits 77% similarity to the castor bean
cysteine endopeptidase and cleaves native precursor polypeptides as well as synthetic oligopeptides like the castor
bean enzyme (Shimada et al, 1994).
A similar endopeptidase was purified from mature
jack bean, Canavalia ensiformis L. (Abe et al., 1993).
Oligonucleotides derived from partial N-terminal amino
acid sequence of the enzyme have been used to obtain
four different cDNA clones which suggests that isoenzymes exist which are encoded by a multigene family
(Takeda et al., 1994). Since similar Asn-specific cysteine
endopeptidases are present in various legumes it was
named legumain (Ishii, 1994; Takeda et al., 1994) according to the current edition of Enzyme Nomenclature (EC
3.4.22.34). A method was published which now permits
simple and sensitive quantitation of corresponding
enzyme activities even in crude extracts (Cornel and
Plaxton, 1994) using benzoyl-L-asparagine-/7-nitroanilide
as the substrate with subsequent diazotization of the
generated p-nitroanilide which makes the spectrophotometric measurement of this reaction product more
sensitive.
Polymorphism of an Asn-specific 33-33.8 kDa vacuolar
11S processing enzyme has also been reported for a
cysteine endopeptidase preparation from mature soybean
(Muramatsu and Fukazawa, 1993). The three isoforms
were isolated which had isoelectric points of 4.94, 4.89,
and 4.85, respectively. They specifically cleaved proglycinin, which was recombinantly produced in E. coli, into
the mature a- and _$-chains at the C-terminus of Asn in
the precursor. Glycinin is the legumin-like US storage
globulin from soybean and the endopeptidase was called
'maturation enzyme'. Whereas the Asn-specific cysteine
endopeptidases reported so far do not seem to be glycosylated, Scott et al. (1992) prepared a glycosylated US
globulin maturation endopeptidase from soybean. The
enzyme which in electrophoresis under denaturing conditions gave 3 polypeptides with relative molecular weights
of 85, 63 and 23 kDa in vitro correctly processed prolegumin from Vicia faba L. as indicated by N-terminal
microsequencing of the cleavage product. Octapeptide
substrates were synthesized with an Asn in the P-position
of the internal cleavage site, and several mutations of the
Asn-residue were produced. Only octapeptides having
Asn in an internal P-position were cleaved by the enzyme.
This confirms its strict Asn-specificity.
Several lines of evidence indicate that this new class of
Asn-specific cysteine endopeptidases is the prime candidate for the propolypeptide processing proteinase that
catalyses the maturation of US and 2S storage proteins
in dicotyledonous seeds. (1) The enzyme has been local-
611
ized to the same compartment as the storage proteins,
namely the vacuoles (developing protein bodies) of cells
in storage endosperm and cotyledons of developing seeds,
(2) in vitro cleavage of the corresponding storage protein
precursors occurs as expected, and (3) the time-course of
the appearance of the corresponding enzyme polypeptide
as well as the time-course of its activity correspond with
the period of biosynthesis and processing of the presumptive in vivo subtrates in developing castor bean (Cornel
and Plaxton, 1994; Hiraiwa et al., 1993). Nevertheless,
conclusive in vivo evidence for the suggested endopeptidase function is still lacking. More conclusive evidence
could be provided if it is possible to inhibit storage
protein maturation by expression of appropriate antisense-DNA constructs of the proteinase in the developing
seeds of transgenic plants.
Legumin processing by limited proteolysis in vitro and
in vivo: An in vitro processing assay for proglycinin of
soybean has been developed by combining the in vitro
transcription/translation of US globulin propolypeptides
followed by oligomer reconstitution (Dickinson et al,
1987, 1989) with an in vitro cleavage assay using the
soybean US globulin maturation enzyme (Scott et al,
1992; Jung et al, unpublished). Prolegumin B from faba
bean which was produced in vitro and could be assembled
into trimers was purified by isopycnic sucrose density
centrifugation and used as substrate in the cleavage assay
(Nielsen et al., 1995). The results of these experiments,
which at least in part are still unpublished, indicate that
only correctly formed prolegumin trimers undergo the
single site cleavage of the peptide bond linking the a- and
jS-chain in the precursor and are thereby converted into
the hexamers. Prolegumin monomers are degraded probably because they assume a conformation where the many
other Asn-flanked sites in the polypeptide are not protected against attack by the maturation enzyme. This
result also suggests that no disassembly of the prolegumin
trimer should take place in the process of trimer to
hexamer transition in vivo.
Confirmation of these in vitro results have come from
tobacco transformation experiments. Prolegumin without
the C-terminal Asn of the a-chain could not be cleaved
in transgenic tobacco seeds and formed only trimers but
no hexamers (Jung et al, unpublished). Prolegumin chain
fragments of various length were fused before the complete chloramphenicol acetyl transferase polypeptide
(CAT). Stable a-chains were formed inside vacuoles if
the constructs contained an intact a//8-chain cleavage site.
The detached fragments comprising partial jS-chain
sequences or the complete ^-chain fused to CAT were
degraded. This suggests that these fusion polypeptides
were recognized as misfolded by the vacuolar proteases.
Legumin-CAT fusions with fragments shorter than the
complete a-chain did not contain the a/$-cleavage site.
612
MOntz
They remained outside the vacuole and were not degraded
(Jung et al., 1993). This processing of prolegumin-CAT
fusions could also be demonstrated in vitro. The enzyme
which catalyses the correct processing as well as the
enzyme responsible for the degradation of the misfolded
chains should be located within the vacuole. Both activities can probably be attributed to the same Asn-specific
cysteine endopeptidase.
The prolegumin processing activity has been localized
to vacuoles of storage tissue cells of developing pumpkin
and castor bean seeds (Hara-Nishimura et al., 1987;
Harley and Lord, 1985). The Asn-specific processing
cysteine endopeptidase has been purified from protein
bodies of castor bean endosperm (Hara-Nishimura et al,
1991). Immunohistochemical studies of this protease
have localized it to the vacuoles of similar cells (HaraNishimura et al, 1993a). Prolegumin trimers, but no
hexamers, are present in the ER of developing storage
tissue cells in pea (Chrispeels et al., 1982), soybean
(Barton et al., 1982) and pumpkin cotyledons (HaraNishimura and Nishimura, 1987). Conversely, only negligible amounts of prolegumin have been found in vacuoles
of corresponding cells in which legumin hexamers predominate. All these results indicate that prolegumin trimer
assembly takes place in the ER. The trimers are then
transferred into the storage vacuoles where they are
cleaved by the processing enzyme. This cleavage of the
a//?-chain peptide linkage transforms the prolegumin
trimer directly into the mature legumin hexamer which is
then able to form deposits in the developing protein
bodies. Formation of the processing enzyme as well as of
the prolegumin are subject of strict developmental control
(Hara-Nishimura and Nishimura, 1987, 1993a). Both
concomitantly undergo intracellular protein transfer
through similar compartments. Since no cleavage of the
prolegumin seems to occur before reaching the vacuole,
the protease should be inactive during its passage through
the secretory pathway and becomes activated after its
arrival in the storage vacuole. A finely tuned interplay of
changing structure function relations between substrate
and enzyme proteins, of enzyme activation and compartmentation is responsible for regulating this process.
Processing of a protein body membrane protein
precursor: Recently it has been shown (Inoue et al, 1995)
that protein body membrane polypeptides MP27 and
MP32 arise from the translation of an mRNA into a
common precursor which must be post-translationally
processed into the two polypeptides. The putative cleavage site in the P-position is flanked by an Asn-residue.
The authors suggest that here also an Asn-specific cysteine
endopeptidase may be the processing enzyme, since, as
above, the precursor polypeptide enters the secretory
pathway and the polypeptides are associated with the
inner surface of the protein body membrane.
Limited proteolysis of provicilin in vacuoles: Posttranslational cleavage of vicilin precursors has been
demonstrated in developing pea (Gatehouse et al., 1981,
1982, 1983) and field bean cotyledons (Scholz et al.,
1983). Some of the polymorphic 50 kDa pea vicilins
contain two internal cleavage sites flanked by Arg and
Lys residues, whereas others are free of such sites. In
mature pea cotyledons sets of distinct polymorphic cleavage products of 33, 19, 16, and 13.5 kDa have been found
which are generated by cleavage at both or only one of
the two preformed sites. The 16 kDa fragment was glycosylated. In field bean 50 kDA vicilin subunits predominate
that are free of such cleavage sites. The proportion of the
vicilin subunits that were cleaved to sizes comparable to
those found in pea was strongly dependent upon the
preparation method. Since in the same legume plant
cleavable as well as non-cleavable forms of 50 kDa vicilin
subunits co-exist and in certain legumes, like garden
beans, no cleavage of the vicilin-homologous phaseolin
subunits takes place, it is difficult to assign a function to
this processing step.
This type of limited proteolysis does not appear to
occur in the processing of convicilin-like subunits of 7S
globulins. Nevertheless, these proteins are subject to
specific processing. In developing cotton seeds (Gossypium
hirsutum L.) the vicilin-like storage proteins, named aglobulins, are represented by two groups of polymorphic subunits, the glycosylated 52 kDa and the nonglycosylated 46.5 kDa polypeptides (Dure and Chlan,
1981). Both are generated by processing of a family of
propolypeptides with molecular weights of about 67 kDa
(Chlan et al., 1986, 1987). An N-terminal 20.5 kDa
fragment is detached by limited proteolysis from the
precursor to form the mature vicilin-like chains. The
putative cleavage site is flanked by Arg-residues, so that
a processing enzyme different from the prolegumin
processing endopeptidase must be responsible. The intracellular site of this processing event still remains to be
elucidated.
Other processing enzymes? A putative aspartate endopeptidase has been partially purified from extracts of developing and mature Brassica napus seeds and used in
processing experiments with in vrtrosynthesized and
radioactively labelled rape napin-like 2S albumin propolypeptide from Arabidopsis thaliana as a substrate (compare
Fig. 1). In addition, synthethic substrates have been used
that had been designed according to the peptide which
links the small and large chain in the albumin precursor
and is excised during its processing (D'Hondt et al,
1993). Internal peptide bonds were cleaved in these
substrates which suggests that the enzyme could also
process the precursor. The propolypeptides were split into
fragments that correspond to the size of the small and
large chains of mature 2S albumin. Thus the authors
Proteolysis of seed storage proteins
claim that they have found a processing protease which
at least in vitro might correctly process 2S storage albumin
precursors of cruciferous seeds. No evidence was presented that the enzyme is present in a compartment where
2S proalbumin processing might take place and how the
N-termini of the mature chains might be generated by
this or an additional enzyme.
Limited proteolysis and storage globulin
degradation in germinating seeds
In mature dry seeds storage proteins are present in the
embryo axis as well as in the storage tissues proper, like
the endosperm, for example, in castor beans, or the
cotyledon mesophyll, for example, in legumes, oil seed
rape, sunflower or pumpkin. However, in the embryo
axis, time-course, pattern and mechanism of storage protein degradation as well as its contribution to nitrogen
supply for the developing embryo and its regulatory
interaction with the major protein degradation processes
in the proper storage tissues has not been investigated.
Research has been focused on proteolytic enzymes and
storage protein degradation in the proper storage tissues.
There, the beginning of measurable storage protein
degradation can be detected at days 2-3 after the start
of imbibition (dai) depending on the species under investigation. Further storage protein breakdown proceeds
much more rapidly in the cotyledons of germinating
Vigna radiata (L.) Wilczek. (Baumgartner and Chrispeels,
1979) or Phaseolns vulgaris L. (Nielsen and Liener, 1984;
Boylan and Sussex, 1987) where 7S globulins strongly
predominate, than in seeds of Pisum sativum L. (Basha
and Beevers, 1975), Vicia faba L. (Lichtenfeld et al.,
1979, 1981) or Glycine max (L.) Merr. (Wilson et al.,
1986) in which nearly 50% or more of the storage protein
is made of 1 IS globulin. Nevertheless, the overall pattern
of storage globulin degradation is similar and, in parallel
to storage globulin breakdown, protein bodies start to
fuse and large vacuoles are regenerated in the storage
tissue cells.
Pattern of storage protein cleavage
If globulins from field beans were analysed by electrophoresis under non-denaturing conditions or by immunoelectrophoresis slight mobility changes were observed for
vicilin and legumin during the first days of germination.
Changes in mobility in immuno-electrophoresis may be
due to changes in the charge (Lichtenfeld et al., 1979).
Similar observations were made with germinating vetch
seeds (Shutov and Vaintraub, 1973), beans (Boylan and
Sussex, 1987) and buckwheat (Dunaevski and Belozerski,
1989a). If samples from early germination stages were
analysed under denaturing conditions, cleavage products
were apparent. As germination progressed, the original
613
bands became weaker and distinct breakdown products
appeared. Taken together these results suggest that limited
proteolysis plays an important role in initiating storage
globulin degradation. After limited proteolysis the holoproteins nearly retain their original electrophoretic mobility under non-denaturing conditions. The holoproteins
also retain their sedimentation characters in sucrose gradient centrifugation. Both results indicate that at this stage
the proteins are largely intact. Only very small fragments
are lost at this stage. Consequently, the amount of
liberated amino acids must be small. This is reflected in
the slow initial decrease of protein nitrogen that was
observed in germinating seeds. Whereas distinct large size
cleavage products were found the small ones have never
been electrophoretically demonstrated, indicating that the
half-lives of the smaller fragments may be much smaller
than those of the large fragments. The amino acids
produced by the rapid breakdown of the small fragments
are transferred to the growing germling where they provide the starting material for new protein biosynthesis.
The breakdown of the major amount of storage globulins
occurs from 4-8 dai depending on the plant species and
it coincides with the major activity of proteolytic enzymes.
Pattern of legumin degradation: In seeds containing both
legumins and vicilins the degradation of legumin proceeds
more slowly (Lichtenfeld et al., 1979, 1981; Wilson et al.,
1986). The a-chain of legumin starts to be degraded first
whereas the jfl-chain remains intact until 5-6 dai in
cotyledons of germinating field bean and soybean when
the major storage protein degradation occurs. This might
be attributed to the topographic position of this chain in
the holoprotein. The a-chains are thought to be exposed
at the surface while the _/8-chains are located inside the
holoprotein. This is supported by two observations. (1)
Antibodies raised against the holoprotein predominantly
react with a-chains, but only weakly or not at all with
the jS-chains in immunoblots; and (2) in short-time
in vitro incubation experiments endopeptidases nearly
exclusively cleave a-chains, but not jff-chains. The major
cleavage intermediates exhibit relative molecular weights
of 24 000 to 30 000 which is larger than the molecular
weight of j9-chains, thus indicating that these intermediates must have been generated from the a-chains.
No detailed analysis of the cleavage products such as
partial N-terminal amino acid sequencing has so far been
performed. Therefore, no exact localization of the cleavage sites is known. In cleavage experiments with trypsin
distinct fragments of the a-chain of glycinin, the leguminlike globulin from soybean, are generated (Shutov et al.,
1993). The fragment sizes suggest that the sites that are
attacked correspond to loops II and 12 in the predicted
secondary structure of legumin (Shutov et al., 1995).
Similar sites should represent the targets of the first,
limited, endopeptidolytic attack during legumin degradation in germinating seeds.
614 MQntz
Pattern of vicilin degradation: Conglycinin is the major
vicilin-like 7S globulin in soybean. During germination
the large a- and a'-subunits of ^-conglycinin disappear
first (Bond and Bowles, 1983; Wilson et al, 1986).
Whereas Bond and Bowles (1983) already found degradation of these polypeptides in mature and imbibing seeds,
Wilson et al. (1986) have reported that the breakdown
of these ^-conglycinin subunits starts at the earliest 1 d
after imbibition. As the a- and a'-^-conglycinin polypeptides disappear, a 51 200 kDa polypeptide accumulates
which reacts with jft-conglycinin-specific antibodies and is
generated by the detachment of an N-terminal fragment
from the large ^-conglycinin subunits (Qi et al., 1992).
This limited proteolysis step resembles the processing
reported for large vicilin subunits of cotton during seed
maturation (Chlan et al., 1986). The intermediates of aand a'-jS-conglycinin degradation start to disappear at 6
dai when new jS-conglycinin-specific intermediates with
mol. wt. 25-31 kDa emerge. The 50 kDa _/?-^-conglycinin
chain does not seem to be degraded until 6 dai. Its
degradation starts at the same time as that of the 51.2 kDa
intermediate degradation product of the a- and a'-ficonglycinin. Therefore, it has not been possible to determine the precursors of the 25-31 kDa conglycinin cleavage products. The degradation of j0-j?-conglycinin
coincides in time with the breakdown of the jfl-chains of
glycinin, the legumin-like US globulin of soybean.
In Vigna radiata and Phaseolus vulgaris, in which the
major globulins are 7S trimers containing predominantly
50 kDa subunits, globulin degradation takes place around
the 3rd to 4th dai (Baumgartner et al, 1979; Nielsen
and Liener, 1984; Boylan and Sussex, 1987). Groups of
degradation intermediates with Mt 20-30 kDa appear.
The non-glycosylated 45.5 kDa phaseolin polypeptide
seems to be degraded more easily than the glycosylated
51 and 48 kDa subunits (Nielsen and Liener, 1984).
Germination proteases
By applying different protease inhibitors to germinating
castor beans (Alpi and Beevers, 1981) and to extracts
from germinating garden beans (Nielsen and Liener,
1984) and mung beans (Chrispeels and Boulter, 1975) it
was possible to determine that sulphydryl proteinases or,
as they are named today, cysteine endopeptidases, are
primarily responsible for storage globulin degradation in
vivo. Numerous publications in which increases in amount
and activity of cysteine endopeptidases are correlated
with the major breakdown of storage globulins, support
this conclusion (for reviews see Wilson, 1986; Shutov and
Vaintraub, 1987; Vierstra, 1994). Some authors also
report the involvement of serine endopeptidase
(Mitsuhashi et al, 1986; Qi et al, 1992) or metalloendopeptidase (Belozerski et al, 1990; Elpidina et al,
1991) in storage globulin degradation.
The pioneering work of Chrispeels's group (reviewed
by Baumgartner and Chrispeels, 1979) on the proteolytic
breakdown of vicilin in germinating seeds of Vigna radiata
has demonstrated that cysteine endopeptidases synthesized de novo, in their case a vicilin peptidohydrolase
(Baumgartner and Chrispeels, 1977), catalyse globulin
degradation in the cotyledons of germinating legume
seeds. Purified vicilin peptidohydrolase has a mol. wt. of
23 kDa, an IEP at pH 3.75 and exhibits maximal activity
at pH5.1. The enzyme preferentially cleaves synthetic
esters with asparagine and glutamine, readily degrades
vicilin in vitro, and exhibits its major activity increase in
germinating seeds from 2-5 dai when most of the vicilin
disappears. It is synthesized at membrane-bound polysomes. The enzyme protein undergoes vesicular transport
into the protein storage vacuole where it initiates the
degradation of globulin (Baumgartner et al, 1978).
Although a protease inhibitor with in vitro activity against
the vicilin peptidohydrolase is present in the cotyledon
cells of germinating Vigna radiata seeds, it is located in a
different subcellular compartment so that it probably
plays no role in the regulation of vicilin degradation
(Baumgartner and Chrispeels, 1976; Chrispeels et al,
1976; Chrispeels and Baumgartner, 1978). Since isolated
protein bodies from mature seeds showed only negligible
endogenous protein degradation and storage protein
breakdown was elicited by the addition of extracts from
4 dai seeds, the de novo synthesis of the vicilin peptidohydrolase is assumed to be the key event in vicilin degradation
(Harris and Chrispeels, 1975; Baumgartner et al, 1978).
This was confirmed by detailed studies on vicilin and
legumin degradation in germinating vetch, Vicia sativa L.
(for review see Shutov and Vaintraub, 1987). In this
system two proteases participate in globulin degradation:
a triggering cysteine endopeptidase A with an Afr 21 kDa
(cDNA-derived mol. wt. 25 244 kDa), which is active
against globulins prepared from mature dry seeds, and a
cysteine endopeptidase, termed proteinase B, with Mr
38 kDa that exhibits strict Asn cleavage specificity. Both
enzymes appear to be absent from the mature seed and
to be de novo synthesized during germination. Since the
substrate for proteinase B is globulins that have already
been modified by the 'triggering' proteinase A, its activity
is only apparent after proteinase A has become active. A
serine endopeptidase appears to be required for the partial
hydrolysis of the large vicilin-like subunits of soybean ficonglycinin, the a- and a'-jS-conglycinin chains (Qi et al,
1992). This has confirmed an earlier study that implicated
a serine endopeptidase in the limited proteolysis of a
64 kDa subunit in cotyledons of germinating Vigna mungo
seeds (Mitsuhashi et al, 1986). In both cases no corresponding enzyme activity could be measured in extracts
from mature dry seeds.
A different model for the initiation of globulin breakdown has been developed by Belozersky and his
Proteolysis of seed storage proteins
group by studying germinating buckwheat (Fagopyrum
esculentum L.). They found a Zn-dependent metalloendopeptidase that forms an inactive complex with an
endogenous protease inhibitor inside the protein bodies
of mature seeds (Belozerski et al, 1990; Elpidina et a!,
1991). The enzyme was shown to degrade in vitro a
legumin preparation from mature buckwheat. They proposed that the dissociation of the enzyme from the
inhibitor which is mediated by zinc ions, triggers globulin
breakdown. In addition, a cysteine endopeptidase has
been isolated from germinating buckwheat which
degrades legumin prepared from seedlings, but not that
prepared from dry seeds (Dunaevsky and Belozersky,
19896). The authors proposed that limited proteolysis of
legumin by the metallo-endopeptidase, which is already
present in mature seeds, forms a prerequisite for further
degradation of legumin by the sulphydryl endopeptidase
and that the activity of the latter protease is feedback
inhibited by degradation products.
Papain-like cysteine endopeptidases: proteinase A: Papainlike cysteine endopeptidases represent the major storage
protein degrading enzymes which appear during seed
germination, for example, in cotyledons of Phaseolus
vulgaris (Boylan and Sussex, 1987), of Vigna mungo,
where it is called SH-EP (Akasofu et al, 1989, 1990) as
well as of Vicia sativa (Becker et al, 19956), where the
enzyme is named proteinase A. A more distantly related
cysteine endopeptidase protein, termed P34, has been
found in the cotyledons of developing, mature and germinating soybean, Glycine max (Kalinski et al, 1990,
1992) although evidence is still lacking that it is enzymatically active. A cysteine endopeptidase from Vicia faba
also seems to belong to this papain-like class of proteinases (Yu and Greenwood, 1994). Only very late
during germination it reaches maximum activity after
approximately 50% of the stored proteins had already
disappeared. A member of this enzyme group has even
been found in pods of developing Phaseolus vulgaris
fruits. It shows more than 90% sequence similarity to
the Vigna mungo SH-EP (Tanaka et al., 1991, 1993).
Homologous storage protein degrading endopeptidases
have been found in other dicotyledonous seeds as well as
cereal grains, like barley and rice, during germination.
Proteinase A, SH-EP, and P34 are synthesized at the
rough endoplasmic reticulum (rER) and eventually
appear in the protein storage vacuole. The primary translation products have mol. wts similar to or larger than
43 kDa and undergo four to five limited proteolytic
cleavage steps (Fig. 2). The first step is the detachment
of the N-terminal signal peptide during translation which
yields proproteinase A, a proteinase A precursor polypeptide, that is comprised of an N-terminal propeptide of
approximately 120 amino acid residues, and a fragment
which corresponds to the mature enzyme. The N-terminal
615
propeptide is detached by two to three consecutive cleavage steps. Whereas the proproteinase SH-EP and its first
cleavage product are inactive, the product of the next
cleavage exhibits very low activity. The enzyme becomes
fully activated by the last proteolytic processing step(s)
(Mitsuhashi and Minamikawa, 1989). The tetrapeptide
KDEL which is present at the C-terminus of cDNAderived sequences of SH-EP and proteinase A precursors
is known to be a ER retention signal. It is lacking in
isolated-mature SH-EP (Okamoto et al., 1994) and proteinase A (Becker et al., 19956) of germinating seeds.
One could speculate that this step coincides with the
transfer of the proenzyme from the ER to the vacuole
and/or an intermediary compartment on the way to the
vacuole. The mature enzymes have different molecular
weights: SH-EP, 33 kDa (electrophoretically determined);
P34, 28.6 kDa; and proteinase A 25.4 kDa (both calculated from cDNA-derived sequences). Exactly where in
the cell the N-terminal propeptide fragments are detached
is still unknown. However, for several reasons discussed
later, at least the final step should take place in the
vacuole. Only the propeptide regions of the precursors of
proteinase A and P34 contain glycosylation sites, which
agrees well to the finding that the mature enzymes are
not glycosylated. The P34 precursor has been shown to
be glycosylated (Kalinski et al, 1992). SH-EP also contains potential gJycosylation sites in the mature polypeptide, but it is not known whether they actually bear
carbohydrate side chains. Papain-like cysteine endopeptidases exhibit optimal activity at pH values near 5. The
mature enzymes have 7 cysteine residues and contain
conserved elements which are known to be part of the
active centre of papain (Fig. 2a). Whereas only a single
gene appears to code for the Vigna mungo SH-EP
(Yamauchi et al., 1992), small gene families with at least
two members have been shown to code for proteinase A
(Becker et al., 19956) and P34 (Kalinski et al., 1990).
Proteinase P34 not only in its primary structure is less
related to SH-EP than proteinase A, but it also has no
C-terminal KDEL-signal for ER-retention. In mature
soybeans P34 forms dimers which are thought to be an
inactive state of the enzyme (Herman et al, 1990; Kalinski
et al., 1992). A Cys residue at position 10 seems to be
responsible for dimerization. In germinating seeds an
N-terminal fragment is detached which transforms P34
into P32. It was speculated that the dimer dissociates by
this step into monomers and the enzyme is activated by
monomer formation. No de novo formation has been
shown during germination.
Other papain-like cysteine endopeptidases, termed
CPR1 and CPR2, have been found in the cotyledons of
germinating Vicia sativa seeds (Becker et al., 1994). They
are formed de novo during germination. The greatest
sequence homology so far found for CPR2 is to a drought
stress-inducible pea cysteine endopeptidase that has been
616
Milntz
found in vegetative organs (Guerrero et al., 1990) and to
a related enzyme from developing soybean cotyledons
(Nong Van Hai et al, 1995). The vetch seed enzyme can
also be induced by drought stress (Fischer, unpublished).
It is still unknown whether these vetch endopeptidases
participate in the storage protein degradation or not.
However, they become active at the time of storage
globulin degradation, and, since the cDNA-derived amino
acid sequences contain N-terminal signal peptides, they
are probably located in the proper subcellular compartment. Sequence comparison with N-terminal sequences
of homologous enzymes suggest that both cysteine endopeptidases contain propeptides that are processed to yield
the mature enzyme. Calculated molecular weights of
mature CPR 1 and 2 are 26.1 and 24.9 kDa, respectively,
and both have activity maxima in the acidic pH-range.
The conservation of elements of the active centre as well
as the positions of the Cys-residues indicate that the
enzymes are related to papain (see also Fig. 2a).
Proteinase B: a legumain-like cysteine endopeptidase:
Proteinase B has been purified from cotyledons of germinating Vicia sativa and partially sequenced. The sequences
were then used to derive oligonucleotides in order to
clone and sequence the corresponding cDNA (Becker
et al., 1995a). The derived amino acid sequence of the
enzyme indicates that it belongs to a class of cysteine
endopeptidases, termed legumain (already referred to),
that have recently been found in maturing castor bean
and soybean as well as in mature jackbean. The enzyme
strictly cleaves at sites with Asn or Asp in P-position and
processes prolegumin like the legumains from developing
seeds. Its pH optimum is 5.6. As is true of legumain from
maturing seeds, the enzyme is synthesized as a precursor
composed of an N-terminal signal peptide (putatively 24
amino acid residues long), followed downstream by a 24
residues N-terminal propeptide, the sequence of the
mature enzyme and finally a 10-12 kDa C-terminal propeptide (Fig. 2b). To generate the mature enzyme, which
has a mol. wt. of 37-39 kDa, the precursor has to undergo
at least three proteolytic processing steps: the cotranslational signal peptide detachment, and the cleavage of the
N-terminal and of the C-terminal propeptides, which are
flanked by Asn residues at the cleavage sites. There are
some indications that the precursor undergoes autocatalytic cleavage and activation. Two potential glycosylation
sites are present in the mature enzyme. Proteinase B is
synthesized de novo during vetch seed germination.
Serine endopeptidase in germinating soybean: The serine
endopeptidase Cl (Qi et al., 1992, 1994) of soybean which
partially cleaves a- and a'-jff-conglycinin chains as well as
the homologous convicilin from pea has a pH optimum
at 3.5 to 4.5 and a mol. wt. of 70 kDa. Although no
corresponding activity could be measured in extracts from
mature seeds, the enzyme as well as jS-conglycinin
degradation could already be detected after 1 d of imbibition. Distinct cleavage products with mol. wt. 48-50 kDa
are generated in a stepwise fashion. The enzyme cleaves
in vitro inside specific clusters of acidic amino acid residues
independent of whether these are present in large vicilin
subunits or in the C-terminal region of the a-chain of
legumin (Qi et al., 1994). A presumably related enzyme
has been found in germinating Vigna mungo seeds
(Mitsuhashi el al., 1986).
Metallo-endopeptidase in germinating buckwheat: A Zncontaining enzyme with a pH optimum between 8.0 and
8.2 has been purified to homogeneity in electrophoretic
analysis. Its relative mol. wt. has been determined to be
34 kDa by electrophoresis under denaturing conditions
and 39 kDa by gel chromatography. The enzyme cleaves
peptide bonds in oxidized insulin 5-chains which contain
Leu, Tyr or Phe in the P-position (Belozerski et al., 1990)
and cleaves legumin prepared from mature buckwheat as
well as from soybean seeds. The metallo-endopeptidase
appears to be reversibly inactivated by association with
an endogenous proteinase inhibitor polypeptide. It has
been localized in protein bodies (Elpidina et al., 1991).
Protein processing by limited proteolysis and the control of
storage protein maturation and breakdown in legume seeds:
an hypothesis
Endopeptidase-specific antibodies were used along with
the appropriate cDNA probes to detect the presence of
the enzyme and mRNA in storage tissues of developing
and germinating seeds and to follow the time-course of
their appearance. Proteinase A- (Kalinski et al., 1992;
Becker et al., 19956) and proteinase B-specific polypeptides (Hara-Nishimura et al, 1993a; Becker et al., 1995a;
Okamoto et al, 1995) have so far been detected in
developing as well as in mature and germinating seeds of
different legumes, castor bean and pumpkin. Polypeptides
with the size of mature proteinase B were found in protein
bodies prepared from vetch seeds 8 h after the start of
imbibition. Precursors of proteinase A could only be
detected in fractions that did not contain protein bodies
and they corresponded in size to precursor polypeptide
bands found in developing seeds. Three days after imbibition, when both these polypeptide bands were no longer
detectable on Western blots, first newly formed proteinase
B mRNA appeared, closely followed by the appearance
of proteinase A mRNA. No signals for proteinase A- and
B-specific mRNAs have been detected at earlier germination days. Approximately 24 h later, the proenzyme bands
appeared in Western blots and these were followed by
the appearance of intermediate precursors as well as those
of mature proteases within the next few days. This
reinforces previous results which indicated that these
proteases are synthesized de novo during germination.
Proteolysis of seed storage proteins
The proteinase B polypeptides that have been detected in
the cotyledons of mature, early imbibing and early germinating seeds must have been synthesized, processed
and transported to protein bodies during seed development (Hara-Nishimura et al., 1993a). On the other hand,
only precursor polypeptide bands of proteinase A have
been found in extracts prepared from maturing vetch
cotyledons and these were absent in protein body fractions
of 8 h imbibed seeds. This indicates that processing and
transport of proteinase A precursors occur either very
late during seed maturation or early after imbibition. This
would make sense, if as presumed by Kalinsky et al.
(1992) and others (Becker et al., 19956) proteinase B
participates in the molecular maturation of proteinase A
precursors which have the necessary Asp residue in the
P-position at the processing site in front of the N-terminus
of the mature polypeptide. Thus, proteinase A, which can
cleave mature globulin holoproteins, would remain outside the protein bodies until storage protein degradation
is induced. Proteinase B, which can not attack mature
globulins, can be safely stored along with the globulins
inside the protein bodies. During protein body development its activity is necessary for the maturation of prolegumin (Mtlntz et al., 1993), 2S protein precursors
617
(Hara-Nishimura et al., 1991, 1993ft) and membrane
proteins (Inoue et al., 1995) at their Asn-flanked cleavage
sites. In a similar way it could contribute to the final
activation of proteinase A precursors during seed imbibition when these precursors are transferred into the protein
bodies. Provided proteinase A-like precursors, which have
been found outside the protein bodies in developing and
mature seeds, have a C-terminal KDEL as found in
similar enzymes of germinating seeds, this tetrapeptide
may be required to delay its transfer into the vacuole.
The detachment of this ER-retention signal would permit
the transfer of the precursor into protein bodies where
the last N-terminal propeptide could then be cleaved by
action of proteinase B. If this hypothesis is correct then
KDEL detachment would play an important role in
controlling breakdown of globulin. In contrast to SH-EP
and proteinase A the precursor of P34 has no KDEL at
its C-terminus (Kalinski et al., 1990) and, as expected, it
has been localized in the protein bodies of maturing
soybean (Kalinsky et al., 1992). The metalloendopepidase of mature buckwheat has also been found to be
located inside the protein bodies along with the buckwheat
legumin (Elpidina et al., 1991). This enzyme and P34 are
supposed to be inactive inside protein bodies of mature
PHOTBNBOOY
PROTEIN BOOT
Fig. 3. Hypothetical scheme of compartmentation, processing of a storage protein (12S globulin taken as the example) and cysteine endopeptidase
precursors as well as proteinase activation to integrate the data so far obtained for cotyledon cells of developing, mature and germinating vetch
(Vicia saliva L.) seeds (A), for soybean (Glycine max L. (Merr.) (B), and buckwheat (Fagopvrum esculemum L.) (C). ER, endoplasmic reticulum,
LEG, legumin; KDEL, C-terminal tetrapeptide, presumably acting as ER retention signal.
618
MQntz
seeds. The metallo-endopeptidase of buckwheat has been
found to be inactivated by its binding to a protease
inhibitor (Fig. 3c) that is also present in the protein
bodies (Elpidina et ai, 1991). The P34 dimer that has
been detected in protein bodies of mature soybean
(Fig. 3b) is thought to represent an inactive state of the
enzyme. Transformation of P34 into P32 is suggested to
generate monomers which are assumed to be the active
enzymes (Herman et ai, 1990; Kalinsky et ai, 1992).
Other proteins, together with the storage globulins, that
are located inside the protein bodies have also to be
protected against an uncontrolled degradation by the
proteinase B-like processing enzyme. This protection
could either be due to an insensitive structure, as in the
case of hexameric legumin, or by subcompartmentation
of the storage vacuole.
The amount of proteinase A and B protein, as visualized in Western blots, is much lower in cotyledons of
developing, mature and early germinating seeds than it is
later on during germling growth when most of the globulin
is broken down. According to the model of Shutov and
Vaintraub (1987) proteinase A begins degradation of
globulins by cleaving a small number of exposed surface
sites following the zipper cleavage mechanism. These
cleavages trigger conformational changes that make the
protein accessible for proteinase B and carboxypeptidase
which are both present in protein bodies of mature seeds.
The low ratio of proteases to globulin early in germination
may explain why only limited globulin degradation occurs
at that time. Through the combined action of proteinase
A, proteinase B and carboxypeptidases, the released
globulin fragments might be rapidly degraded down to
small peptides and even amino acids. This would explain
why it has not been possible to detect cysteine endopeptidase activity and cleavage products in extracts from early
germination stages. It is still unclear how important the
stored cysteine endopeptidases are for storage protein
breakdown since they are eventually replaced by large
quantities of de novo synthesized enzymes with similar
specificities. Two findings indicate that they are active.
(1) early changes in mobility of the holoproteins which
have been attributed to charge shifts resulting from the
detachment of fragments containing acid and/or basic
amino acid residues. Clusters of such residues are located
in the N-terminal regions of large vicilin subunits and in
the C-terminal region of a-chains from legumin. Both
regions are known to be cleaved at the initiation of
globulin breakdown. However, these charge shifts have
also been interpreted as an effect of protein deamidation
and recently a plant protein deamidase has been characterized in germinating wheat (Vaintraub et ai, 1992). (2)
Proteinase A (called vignain) and B (called legumain)
activity could be detected in extracts of mature seeds of
Vigna aconitifolia by Kembhavi et al. (1993). Furthermore, much higher activity was measured in extracts of
seeds during the first 3 dai, if the extracts were acidified.
Without acidification the activities of both proteinases
increased from 0 h of imbibition (hai) and peaked 36 hai
with a subsequent decrease until it reached nearly zero
after 72 hai. Proteases of other classes have also been
reported to be present in dry seeds, among them serine
endopeptidases and metallo-endopeptidases (see review
by Wilson, 1986). As mentioned above a serine endopeptidase has been shown to catalyse the initial cleavages of
large vicilin subunits. In this case the de novo formation
of the enzyme appears to be a prerequisite for the
degradation of these polypeptides (Qi et ai, 1992).
According to Belozerski et al. (1990) the buckwheat
metallo-endopeptidase which is assumed to initiate legumin degradation is present in an inhibitor-inactivated
state in the mature seed and it becomes activated by
inhibitor dissociation (Elpidina et al., 1991).
The histochemical analysis of germinating mung bean
(Harris and Chrispeels, 1975) and soybean cotyledons
(Diaz et al., 1993) has revealed that storage protein
degradation does not start synchronously in the whole
storage tissue. It is slowly initiated only in subepidermal
cell layers (mung bean) or in cells adjacent to the vascular
bundles (soybean). From these starting sites, which can
obviously be different in different grain legumes, the
increase in endopeptidase activity as well as in protein
degradation proceeds wave-like inwards through the
tissue. The low levels of proteinase and protein breakdown
activities that have been measured in extracts from seeds
of these early germination stages could result from the
small percentage of cells where protein reactivation has
already been started in storage tissue. Therefore, initially
the small amounts of stored endopeptidases should only
become active in protein degradation inside the protein
bodies of limited cell layers. It can even be speculated
that in these cell layers enzyme de novo synthesis has
already been started. The corresponding mRNA levels
remain below the limits of sensitivity of Northern blots
which always have been done with extracts from a tissue
where the majority of cells still were inactive in transcription of endopeptidase genes. New experimental
approaches are needed to elucidate the mechanisms which
initiate protein reactivation in storage tissues during
imbibition and early germination of seeds.
Protein storage vacuoles have been thought to be
formed by transformation of the vegetative vacuole in
the storage tissue cells which already exists before globulin
deposition starts (Chrispeels, 1991). Recently, it has been
reported that in developing pea cotyledons the protein
storage vacuole is formed de novo (Hoh et ai, 1995;
Robinson et ai, 1995). The vegetative vacuole degenerates. In our laboratory this was also observed in developing cotyledons of Vicia narbonensis L. (Hillmer
unpublished). Protein bodies seem to be generated
from the storage vacuole by two different mechanisms:
Proteolysis of seed storage proteins
(1) budding of the storage vacuole during early maturation
stages of storage protein deposition; and (2) fragmentation
during later stages. In addition, it has been suggested that
protein bodies could be generated directly from the ER
during late maturation (Robinson and Hinz, 1995). Thus,
as suggested previously by Adler and Miintz (1993),
differential protein body formation seems to occur. It
remains to be investigated whether the different protein
bodies are similarly equipped with proteinases or not.
Since small gene families encode proteinases A and B,
differential gene activation in developing and germinating
seeds might lead to the formation of isozymes during the
different ontogenetic periods. Thus according to our
hypothesis (Fig. 3) processing as well as degradation of
storage globulins in the storage tissues of developing and
germinating seeds might be controlled (1) by differential
expression of genes encoding pre-propolypeptides of isoforms of different cysteine endopeptidases, (2) by stepwise
and differential limited proteolysis of these endopeptidase
precursors which finally leads to the generation of the
active mature enzyme in the vacuole; (3) by transient
differential compartmentation of cysteine endopeptidase
precursors and activation-catalysing enzyme(s) as well as
(4) by transient differential compartmentation of degradation-triggering cysteine endopeptidase and its storage
globulin substrate. It is predicted that other endopeptidases contributing to storage globulin processing or
degradation might be controlled in a similar fashion. In
addition, specific conformational changes induced by the
action of the proteases on their substrate globulins
contribute to the control of processing and degradation
of storage proteins. The rate of globulin breakdown may
also be regulated via feedback regulation by the level of
amino acids at the site of storage protein breakdown as
has been established for the buckwheat cysteine endopeptidase (Dunaevski and Belozerski, 1989b). Protein reserve
activation in storage tissues seems to be under the control
of the embryo axis. Cotyledons detached from the axis
before imbibition exhibited no increase in endopeptidase
activity and globulin breakdown. The mechanisms of
signal transduction between axis and cotyledons or endosperm are still under investigation (recently reviewed by
Bewley and Black, 1994). Depletion of amino acids in
the growing axis, which is the major site of protein
biosynthesis in the germinating seed (Dunaevsky and
Belozersky, 1993) as well as phytohormonal signals transmitted from the axis to the storage tissues (Gifford el ai,
1984; Nandi et ai, 1995) have been implicated in controlling the breakdown of protein reserves.
Complete breakdown of storage proteins is the result
of massive de novo proteinase formation and the combined
action of endo- and carboxypeptidases inside the protein
bodies and vacuoles in the storage tissue cells of midgermination seeds. Both amino acids and short peptides
are transported from the vacuolar compartment into the
619
cytoplasm where amino- and dipeptidases degrade them
further. The amino acids are then used to meet metabolic
demands in the storage tissue itself, like the biosynthesis
of several reserve degrading hydrolases, and to meet the
demands imposed by the major site of protein biosynthesis
in the germinating seed and growing germling, the
embryo axis.
Acknowledgements
The author expresses his sincere gratitude to Dr C Becker, Dr
S Hillmer and above all to Dr D Waddel for critically reading
the manuscript which, in addition, has been very much improved
by the language editing done by Dr D Waddell. The skilful
drawing of the figures by Mrs KJlian and the careful arrangement
of the list of references by Mrs. E Bielig are gratefully
acknowledged.
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