Download Amino Acid Uptake for the Synthesis of Secretory Protein by the

Reprinted from J. Nuclear Agrjc. BioI., Vol. 9 (1980) pp. 148-150
Amino Acid Uptake for the Synthesis of Secretory
Protein by the Mammary Gland
N.
C. GA:"lGUL!
National Dajry Research Institute, Karnal-13200 1
ABsrRAcr
With the adveDt of radlotracen, studies with "C· and "H·labelled amino acids provided ovenmelming evidence
that mlJk proteins are of matIlID!lJ'y gland origin. Of the lactoproteins 8)1J1thesi.zed, casein IC't'OIInts for 80 per ceat
or the total proteins. Mammary secretory cells synthesize e.>selltially six In8jof- II1IIJIldW'}"SJI(lclftc proteiJU Demely.
tIS .. .u" ,... K-caseUIs and two odIer wbey )IJ'OUlIs. «Aactat.buoUo ((I·LA) and p.)actogloblaIl.n (p.LG).
A 'metabotk ruohrtion' ocrun in the I1lll1U11ry gla_ dlUUtg lactation regulated by a serifS of NIOOIM:S
at cellUlar level. During ~Il differentiation i-. ~t5t81/0n aud lactation, illCl'ease IR respective JnRNAs loo-ce!l Dlilk pr0tei. synthesis. The proteins wblcll are of secretnry IUltllrC were observed to be prefere.tially synthesLse4 by the bound
ribosome. studded with knob.llke protrusions In the membranes termed 'rougb endoplasmic reticulum', 'Post·translatlow modifications occur during the secretory route of these synthesized proteins from the RER through the Gola!
apparatus and secretory vesicles and its final ejection into the lumen with other milk components.
Using wheat germ cell·free trllllsiation system with mRNA of tbe kind, some interestlni dJJJ'erence between
tbe ap,areot molecular wrl&hts of these in vitro S)'Dtllesized milk proteins from those syotheslzed In "fro, bave bun
wen docammfs in recent years. The coocept or the preaasor protein syntbesls, more gencnJiy termed as 'PretrOl:em'.
ba'in& a N·tmnill3l hydropbobic amino acid u tea<>ioo, lias bem mlbUsW ror as .. " K-caselns ud a·LA. and
~LG, wilb IS additiooal aoooo acid resiclue Cor \lS" ~-a!sdos a nd 11, 18 and 19 re:ddltf! Cor K-caseia. (I·LA
and 8·W, rfSpectlrely. Tills supports the ' Slanal byPOUtesis' of Blobel Cor Pfeproteln syntbesb In lbe uu,lIlIDIlI')'
.......
The lifer Cactor stimulating mammary protein synthesis is now helni studied in in vitro wheat ierDl cell·free
system In the laboratory to elucidate the involvement or non·target tissues like Ufer, if any. in lactation.
Amongst the secretory proteins. milk proteins
occupy an unique positio n being synthesized by the
mammary gland using blood amino acids as
precursors. With the advent of radio tracers, the
incorporation studies with uC and sH·labelled
amino acids in the milk proteins by the mammary
gland have clearly established that these secretory
proteins are of mammary origin. Amongst the milk
J . Nur:letU' Agric. BioI., Yol. 9 (1980)
proteins, caseins are the majo r secretory proteins
synthesized by the lactating mammary gland.
accounting for almost 80 per cent of the total lacto·
protein content. After being synthesized, they arc
excreted as stable aggregrates called casein micelles
containing upto several millinn casein molecules.
Earlier tracer studies have revealed that mammary
secretory cells synthesize essentially six major specific
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proteins namely, (;(Sl' a.SI, ~- and K-casein and two
other, _a._lactalbumin (LA) and ~-lactoglobuliIlS (LG).
These investigations were concerned with the
precursors of blood or mammary origin for milk
proteins.
Events in lactation:
Since the mammary gland is under the influence
of hormones and differentiation during gestation
and lactation, the levels of milk proteins together
with the activities and quaali[ies of their respective
mRNAs differ during the dil\"e rentiation sequence.
Three to 4-fold increament over the virgin level,
was observed in both a.-LA and casein during 1st
day of gestation. The levels of cr;-LA mRNA l\nd
casein on RNA increased by abo ut 8- and 6-fold,
respectively, during first week of gestation and increase further until parturition. During lactation,
these levels increased until 8th to 12th day.
Role of 'Rough endoplusmic reliculum' :
There have been recently exciting developments
in our understanding of the way in which proteins
synthesised in the intact mammalian cell are directed
towards specific subcellular and especially, extracellular locations. The mammary secretory ceU is
an example where a few wcll·defined proteins are
synthesized in large quantities for export. The
electron microscopic studies revealed that in secretory
cells, such as those of Liver, pancreas and m.lnlmary
gland, there is an extensive system of membranes,
termed endoplasmic reticulum. much of which is
studded with knob-like protrusions giving it a
'rough' appearance. The protrusions on the "rough
endoplasmic reticulum' (RER) were shown to be
ribosomes. The lactation-specific proteins present
in major amounlS are synthesized in the RER
under genetic control and undergo further posttranslational modifications in their secretory route
from the RER through the Golgi apparatus and
secretory vesicles before ejection into the lumen with
other milk components.
rences betWeen the apparent molecular weights of
these synthesized milk. proteins from those synthesized in vil-o, as isolated from normal milk.
Studies with guinea pig cr;-LA have shown the
synthesis in ri"o of a form containing upto 10
additional amino acids at N-terminal end. 1t apparently represents a pre-«.·LA form which is then
cleaved in vh'o to give cr;-LA as found in milkl.
The existence of a precursor protein for cr;-LA, form
the basis of an attractive explanation for the synthesis of the export milk proteins, consistent with
a mechanism postulated from studies on export
protein synthesized in other systelns.
Signal hyporhf'sis ;
This phenomcnon gave birth to the 'signal hypothesis' as proposed by Blobel and Sabatini', who
suggested that the secretory proteins are synthesized
as larger polypeptide chains with transient amino
termina l extensions (the signals), which interact
with RER membranes, thus triggering the binding
of the functioning ribosomes which provide the
topological conditions for the victorial transfer of
nascent chailts across the R ER membrane. Once the
growing chains have started to cross the RER membranes, the signals are selectively removed through
cleavage by a specific membrane-bound protease(s).
All these findings led to the idea that there might
be a general d ifference between the proteins synthesized on membrane-bound and on free ribosomes.
Not only would the synthesis of secretory proteins
be restricted to membrane-bound ribosomes, but
that of intracellular proteins would be restricted to
free ribosomes. Accordingly a large number of
studies were performed 10 identify the site of synthesis of specific protei.ns, usually employing immunochemical techniques to identify the latter. These
provided formidable evidence that ribosomes syn·
thesizing a wide spectrum of secretory proteins are
located atmost exclusively on membranes, examples
being albumin (in liver), immunoglobulins (in plasma
cells) and fl- lactoglubulin (in the mammary gland).
Cell-free translation: A precursor prolein synthesis :
S}'nlhesis of secretory milk proteins:
Epithelial cells in the mammary gland proliferate extensively during pregnancy and synthesize
large amounts of mammary specific proteins, «-LA
and casein-during gestation and lactation. Milk
protein mRNAs have been isolated from various
species with the ultimate objective of understanding
the mechanisms that regulate the synthesis of these
proteins at the level of transcription and tntnslation.
Using a wheat germ translational system it has been
shown that the total RNA of 4 to 5 day lactation
gland synthesizes cr;-LA and total caseins in a ratio
of 1 :25, However, the proportion of the proteins
coded by these messages is markedly different which
suggests post·transcription controls in the accumulation of the proteins.
The use of purified rnRN A preparations coding
for milk proteins in in vitro cell·free protein synthesizing systems has shown some interesting diffe149
Recent studies have further shown that the secre·
tory milk proteins are essentially synthesized on
membrane-bound polysomes and that mRNA s isolated fr om such polysomes are faithfully translated
into lactoproteins by various ceU-free systems derived
from mammalian cells. Recently. workers3. 4 have
analysed by automated Edman degradation the radiolabelled products resulting from the tra_05lation of
ovine mammary mRNAs in a wheat-germ ceU·free
system, :md subsequently separated from each other
by immunoprecipitation. The radio sequence data
clearly demonstrated the occurrence of precursors
of the 6 Illlljor secretory milk proteins. They have
furthe r succeeded in selectively removing the signal
peptides from still growing and completed polypeptide chains with mammary microsomal membranes
and a soluble membrane deoxycholate extract, respectively, thus demonstratil?-g the occurrence of a
mammary membrane-bound proteinase enzyme able
to cleave the signal sequences from nascent prelactoproleins.
The radiolabeUed primary translation prod ucts
of Qvine mammary rnRNAs synth.esized in a wheatgerm cell-free system were observed to have precursors
with amino terminal extension. Such extension in
case of 3 'Ca-sensitive' caseins (a5 \ . IX~ and 13), Kcasein, ~·lactoglobulin and a.-lactalbumin were round
to have 15.21. IS and 19 additional amino acid residues, respectively, at N-terminal end~. The extra
pieces of these various lactoproteins were similar to
'signal" peptides of other secretory proteins in their
length and hydrophobicity. These preprotein in aU
the cases has methionine at its N-terminal end.
Glycosyiatloll :
A useful approach for the investigation of the
temporal and topological relationships between protein biosynthesis and core glycosylation was described
recently. Core glycosylation of a secretory glycoprotein like rat a -lactalbumin, was observed to occur in
the reconstituted system. It demonstrates that core
glycosyJalion of newly synthesized rat «-LA does
occur In l'itro in the microsomal membrane supplemented system. Processing, segregation, and core
glycosyJation were observed to proceed only when
memberanes were present during translation and not
when they were added after translation'.
A fiver-factor:
The presence of a heat-stable protein in the liver
of lactating rabbit and goat, stimulating mammary
protein synthesis ill vitro, has been recently demonstrated in the laboratory. Attempts are now being
made to characterize it further by using mRNA
from lactating and non-lactating liver from these
animals in a wheat germ cell-free translation system.
The appearance of mammary specific proteins during
the lactation cycle is also being monitored using
mRNA and cell-free translation system and by
irnmunoprecipitation of the radiolabelled products
with the final objective of eliciting the role of signal
peptides in the synthesis of mammary-specific proteins.
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Blobel, G. & Sab:uini, O. D., Biomembrones. 2 ( 197 1) 193.
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