Download Do asparagine-linked carbohydrate chains in glycoproteins have a

Bioscience Reports, Vol. 6, No. 8, 1986
Hypothesis
Do Asparagine-Linked Carbohydrate Chains
in Glycoproteins Have a Preference for/ Bends?
Jaap J. Beintema
Received August 18, 1986
KEYWORDS: glycoprotein; conformation; fl-bend.
X-ray structures of the conformation of carbohydrate moieties and connected regions
of glycoproteins are summarized. Evidence is presented that there is some preference
for carbohydrate attachment at/~-bends. Evolution may have favored glycosylation to
occur at bends to ensure free mobility of the carbohydrate moieties.
INTRODUCTION
Although current nucleic acid sequencing generally leads more rapidly to
knowledge of the primary structure of a protein than classical protein sequencing, the
latter remains essential to determine posttranslational modifications of the primary
sequences. One of the most frequently occurring modifications of proteins is Nglycosylation of asparagine residues in Asn-X-Ser/Thr sequences. During an extensive
study of the molecular evolution of pancreatic ribonuclease a large number of
observations have been made on its glycosylation characteristics (1-4). These studies
provided support for the current understanding of glycoprotein structure and
biosynthesis (5).
Only asparagine residues in Asn-X-Ser/Thr sequences may act as attachment sites
for carbohydrate (6). The transfer of an already assembled oligosaccharide moiety to a
nascent polypeptide occurs in the lumen of the rough endoplasmic reticulum before
Biochemisch Laboratorium, Rijksuniversiteit Groningen, Nijenborgh 16, 9747 AG Groningen, The
Netherlands.
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0144-8463/86/0800-0709505.00/09 1986PlenumPublishingCorporation
710
Beintema
final folding of the protein (5). Carbohydrate attachment is thus a co-translational
event, as could have been concluded from the buried position of the threonine side
chain in the Asn-Leu-Thr sequence of bovine pancreatic ribonuclease (1, 7), a minor
component of which, the B form, has carbohydrate attached.
Although carbohydrate is always attached to Asn-X-Ser/Thr sequences, not all
such sequences in proteins synthesized in rough endoplasmic reticulum bear
carbohydrate. Probably, other structural features of the protein determine the extent
of glycosylation of a given site and further processing of the carbohydrate moiety.
Investigations with synthetic peptides (8) and studies of species variants of
glycoproteins (1-4) indicate that amino acid replacements near the Asn-X-Ser/Thr
sequence, and also residue X, affect glycosylation. Also, the capability of the
glycosylating system has a decisive influence. This not only varies with species (1-4),
but also with tissue, as was shown by studies of the extent of glycosylation of
deoxyribonuclease from bovine pancreas and parotid gland (9) and of ribonuclease
from human pancreas, urine and semen (10).
X-ray studies of a number of glycoproteins have been published (Fc fragments of
immunoglobulin molecules, Refs. 11, I2; haemagglutinin of influenza virus, Refs. 13,
14; neuraminidase of influenza virus, Ref. 15; hemocyanin from Panulirus interruptus,
Ref. 16; bovine pancreatic deoxyribonuclease I, Ref. 17). In addition, threedimensional structures are known of non-glycosylated proteins that also occur in a
glycosylated form, or glycosylated close relatives of which occur (e.g. ribonuclease,
Refs. 18-20; Japanese quail ovomucoid third domain, Ref. 21). Their structures may
be compared to derive any rules which the conformation of carbohydrate moieties and
connected regions of glycoproteins must obey.
It has been proposed that asparagine-linked carbohydrate occurs only at external
fl-bends in protein structures (22, 23). However, X-ray structures show a variety of
conformations: carbohydrate is attached to homologous positions in a fl-bend in the
Fc fragments of human and rabbit immunoglobulin (11, 12), and to an asparagine in
the middle of a fl-strand structure in hemocyanin (16), and two of the seven
carbohydrate chains in influenza virus haemagglutinin are attached to helical-type
structures, two to fl-sheets and two to fl-bends, while the conformation of the seventh
site is unknown because of invisibility of the polypeptide chain at that site on the
electron density map (14).
FUNCTION OF ATTACHED CARBOHYDRATE
Although our knowledge of structure and biosynthesis of glycoproteins has
increased considerably during recent years, functional aspects of the attachment of
carbohydrate remain more obscure. Possibly, carbohydrate moieties may have several
functions. One should even consider the possibility that they sometimes have no
function at all, as may be the case with the short oligosaccharide chain in bovine
ribonuclease B, the minor glycosylated component. Thus, if an Asn-X-Ser/Thr
sequence passes the glycosylating enzyme system during biosynthesis of a polypeptide
chain on the endoplasmic reticulum, the protein will usually be glycosylated. This may
be an evolutionarily neutral property or a functionally or structurally advantageous
one. Evolutionary selection against the occurrence of Asn-X-Ser/Thr sequences should
Conformation of Carbohydrate Attachment Sites
711
occur ifglycosylation is disadvantageous for the stability or the function of the protein.
Evidence for this has been presented by Sinohara and Maruyama (24), who observed
that Asn-X-Ser/Thr sequences occur less frequently than random in extracellular
proteins from eukaryotes.
Possible functional roles of carbohydrate moieties are as intracellular or
extracellular recognition signals for other proteins, as specific building elements in the
three-dimensional structures of proteins and as a more diffuse general protection of
protein structures. Depending on the type of function, the mobility of carbohydrate
moieties relative to the protein core and, thereby their visibility on electron density
maps of crystals, may vary. Visible carbohydrate moieties occur in the crystal
structures of both immunoglobulins (11,12), of hemocyanin (16) and of
deoxyribonuclease (17). Only part of them are visible in both influenza virus proteins
(13-15), the other ones and the carbohydrate moiety in kallikrein (25) are invisible.
Inivisibility may be caused either by a high free mobility relative to the core, even in the
crystalline state, or by adoption of a number of different orientations relative to the
core (26).
STRUCTURE OF ATTACHED CARBOHYDRATE
Crystal structures, both of glycoproteins and of oligosaccharides with covalent
structures as encountered in glycoproteins (27), and Nuclear Magnetic Resonance
~[MR) studies (28), indicate that the two internal N-acetylglycosamines and the
innermost mannose linked to them form a hydrogen-bonded rigid coplanar "celluloselike" structure. Thus, the visibility of carbohydrate moieties in crystal structures
should be determined primarily by the freedom of rotation of the side-chain of
asparagine and of the internal trisaccharide core relative to the protein.
In immunoglobulin Fc fragments (11, 12) and in the influenza virus glycoproteins
(13-15), carbohydrate moieties participate extensively in inter-subunit contacts. They
may have an important structural role and, therefore, have a discrete position in the
conformation. One of the other roles of attached carbohydrate in influenza virus
proteins may be masking of certain regions of the protein surface from interaction with
the immune system of the host (14).
The carbohydrate chains in Fc fragments fold back along the surface of the
subunits to which they are connected. This is possible because of the presence of a cisamide bond between asparagine and carbohydrate. A contribution to the
immobilization of a carbohydrate chain connected by a trans-amide bond to virus
haemagglutinin is provided by a hydrogen bond between the C-6 hydroxyl of the
innermost N-acetyiglucosamine and the hydroxyl group of a threonine in the fl-strand,
located two residues from the asparagine to which the carbohydrate is linked (14). In
hemocyanin, the visible carbohydrate chain is also linked to an asparagine residue
in a E-sheet structure. This chain has very little other contact with the protein
surface. However, the density around the asparagine-side chain suggests that its
carbonyl oxygen is hydrogen-bonded to the side-chain of an arginine residue in the
opposite strand of the fl-sheet (Volbeda, A. and Hol, W. G. J., personal
communication). Since steric hindrance restricts rotation around the bond between the
innermost N-acetylglucosamine and the nitrogen of the amide group of asparagine
712
Beintema
rather severely, and since the amide group itself is planar, mobility of the planar
carbohydrate core region relative to the polypeptide backbone may be largely
dependent upon rotation around the two bonds between the C-atoms of the asparagine
side-chain. Thus, hydrogen bonding of the carbonyl group of asparagine should
restrict the mobility of the carbohydrate linked to it.
Recently, Montreuil (29) has reviewed evidence on spatial structures of glycan
chains of glycoproteins and proposed an umbrella-like conformation with the rigid
internal trisaccharide core extending more or less perpendicular to the protein surface
and the branches (antennae) parallel to the surface. The latter may be mobile or make
specific contacts with the protein surface. There is evidence, for instance, that the
~(1 ---, 6)-linked chain may fold back along the core region (29), enlarging the proteincarbohydrate contact area. However, simple-type oligosaccharide chains, such as
occur in bovine ribonuclease B, cannot form an additional interaction site.
ATTACHMENT AT fl-BENDS ?
The strict requirement of carbohydrate attachment for an Asn-X-Ser/Thr
sequence suggests that the polypeptide substrate should have a very specific
conformation--at least temporarily--in order to be recognized by the active site of the
glycosylating enzyme system. A hypothetical structure of such a conformation has
been described by Marshall (6). It is lost during final folding of the polypeptide chain,
for glycoproteins of known structure have different conformations near their
carbohydrate attachment sites (11-17). A change in polypeptide conformation after
posttranslational modification has also been hypothesized for the hydroxylation of
proline in collagen before formation of the collagen triple-chain fold (30).
However, there may be a preference of carbohydrate for a localization at external
bends of protein structure. Carbohydrate attachment has been observed to occur at
positions 21, 22, 34, 62, 76 and 88 in ribonucleases (1, 2, 3, 4, 10). All these positions are
localized in or close to fl-turns or to bends which do not strictly obey to the definition of
a fl-turn in bovine ribonuclease A (20). X-ray structures of other non-glycosylated
proteins also indicate that carbohydrate is attached to asparagine residues in external
fl-turns (21). Aubert et al. (22) and Beeley (23) suggested that carbohydrate occurs only
at asparagine residues in fl-bends. They found a rather high B-turn potential of peptide
stretches with asparagine-linked carbohydrate in a number of glycoproteins with the
method of Chou and Fasman (31). (The Asn-X-Ser/Thr sequence itself has a lower
potential for forming fl-turns.) However, there was no preferential occurrence of
asparagine at any of the four positions in a/?-bend, and if asparagine occurs at the third
or fourth position, the serine or threonine is outside the bend. Therefore, the fl-bend
hypothesis does not fulfil the strict specificity requirement of an enzyme-catalyzed
reaction. Also, Mononen and Karjalainen (32) observed that, when they predicted
secondary-structural elements in a large number of proteins, about 70 ~o of the Asn-XSer/Thr sequences occurred in fl-bends, 20 % in fl-sheets and 10 % in e-helices, and that
there was no difference between glycosylated and non-glycosylated sequences.
Hypotheses on conformational differences between homologous proteins derived
from differences in /3-turn potentia! should also be considered with caution. The
attachment of carbohydrate to the Asn-Gly-Ser sequence in rat e-lactalbumin but not
Conformation of Carbohydrate Attachment Sites
713
to the homologous Asn-Gln-Ser sequence in bovine ~-lactalbumin was explained by
the shift of a #-bend (33). However, a model of the sequence built into the crystal
structure of lysozyme predicted a bend in bovine ~-lactalbumin exactly at the position
of the Asn-Gln-Ser sequence (34).
CONCLUSIONS
Thus, there is a preference, though not an absolute one, for the location of
carbohydrate moieties at external #-bends, especially in relatively small proteins. This
preference may be explained not from structural requirements but from a functional
one, in which attached carbohydrate acts as a diffuse protection layer around the
protein. The carbohydrate layer may be described as part of the surrounding solvent of
these glycoproteins, but protects them from proteolytic degradation or adsorption to
cellular surfaces (35, 1). Also, the carbohydrate layer decreases the ionic strength in the
vicinity of the protein surface (35, 36). The protective function of the carbohydrate
chains is ensured by their free mobility relative to the protein surface, like in the
umbrella conformation proposed by Montreuil (29). Figure 1 presents such a structure
for a ribonuclease molecule with three attached carbohydrate moieties. However, such
mobility is only possible if the asparagine side-chain can rotate freely and if there are no
further interactions between the carbohydrate and the protein surface. This
requirement can only be met if the attachment occurs at external bends. Therefore,
selection has favored glycosylation at positions in nascent polypeptide chains that
adopt this conformation during folding.
Fig. 1. Space-fillingmodels of bovine ribonuclease(left)and of a glycosylated
ribonuclease with three biantennary complex-type oligosaccharide chains
attached to the surface (right). This Figure is a collageof the Figure on p. 81 in
Ref. 37 (ribonuclease) and Figure llB in ~Ref. 29 (spatial conformation of a
biantennary glycan of the N-acetyllactosaminetype in the T-conformation).
Beintema
714
ACKNOWLEDGEMENTS
This paper results from a study together with Dr N. Borkakoti at Birbeck
College (London, UK) to attach carbohydrate to bovine pancreatic ribonuclease by
model building, using an Evans & Sutherland Picture II molecular graphics system.
Many thanks are due to Dr R. N. Campagne for carefully reading the manuscript.
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