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Strategies for the biosynthesis of bioactive peptides
TINS -June 1983
229
Strategies for the biosynthesis of bioactive peptides Richard E. Mains, Betty A. Eipper, Christopher C. Glembotski and Robert M. Dores Neuropeptides and small peptide hormones are usually synthesized as part o f large, inactive precursor proteins. Although the sequences o f many o f these pro-molecules are now known, much remains to be learned about the post-translational processing events that must occur to generate the bioactive products. W i t h the recent d e v e l o p m e n t o f rapid D N A s e q u e n c i n g techniques, o u r k n o w l e d g e o f precursors to s m a l l b i o a c t i v e peptides has g r o w n t r e m e n d o u s l y . For m a n y peptides w e k n o w the s e q u e n c e o f the pre-prom o l e c u l e and the structure o f the final b i o a c t i v e peptide product, but w e h a v e v e r y little k n o w l e d g e o f the i n t e r v e n i n g b i o s y n t h e t i c events. In this r e v i e w w e h a v e c h o s e n to d i s c u s s the f o l l o w i n g s o m e w h a t arbitrary subset o f the m a n y m o d i f i c a t i o n s k n o w n to o c c u r in n e u r o p e p t i d e and peptide h o r m o n e b i o s y n t h e s i s : proteolysis, amidation, acetylation, p h o s p h o r y l a t i o n and sulfation. O t h e r r e v i e w s can b e c o n s u l t e d for discussions of two important modifications that are not c o n s i d e r e d here: the function and r e m o v a l o f s i g n a l peptides and the addition and m a t u r a t i o n o f a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e chains; in addition, w e h a v e not a t t e m p t e d to r e v i e w the vast literature on purification and characterization o f proteolytic p r o c e s s i n g e n z y m e s . W e h o p e to c o n v e y to TINS readers s o m e o f our e x c i t e m e n t about h o w m u c h r e m a i n s to be d i s c o v e r e d about the b i o s y n t h e s i s o f bioactive p e p t i d e s * . Precursors and products S i n c e protein precursors are a l w a y s bigger than their product peptides, proteases play a k e y role in the b i o s y n t h e s i s o f all the
p e p t i d e s to be d i s c u s s e d . S e l e c t e d e x a m ples o f protein precursors and s o m e o f their p r o d u c t s are s h o w n in Fig. 1. Our initial d i s c u s s i o n w i l l be c o n f i n e d to the o v e r a l l d e s i g n o f p r e c u r s o r s and their product peptides. B y o b s e r v i n g i m p o r t a n t patterns in the structural r e l a t i o n s h i p s o f p r e c u r s o r s to their products, w e hope to r e a c h s o m e con* Due to the restriction on the number of references allowed in this review, we have cited only the work most relevant to our limited discussion; there are many other studies that have been key in elucidating the mechanisms of bioactive peptide biosynthesis. In general, we have cited only one or two papers from a research group, literature survey completed August 1982.
c l u s i o n s r e g a r d i n g the proteolytic processing that results in the f o r m a t i o n o f the p r o d u c t peptides. First, the size o f the p r e c u r s o r protein is not s i m p l y c o r r e l a t e d w i t h the size o f the b i o l o g i c a l l y a c t i v e product peptide. For example, methionine-enkephalin (ME) and" l e u c i n e - e n k e p h a l i n ( L E ) are e a c h o n l y five a m i n o acids in length, yet they are d e r i v e d from one o f the b i g g e s t m o l e c u l e s s h o w n in Fig. 1. The b i o l o g i c a l l y active p r o d u c t peptide can o c c u r a n y w h e r e w i t h i n the structure o f its precursor. A r g i n i n e v a s o p r e s s i n ( A V P ) o c c u r s at the e x t r e m e N H ~ t e r m i n u s o f the p r o h o r m o n e , next to the s i g n a l peptide. Gastrin, c a l c i t o n i n , a d r e n o c o r t i c o t r o p i c h o r m o n e ( A C T H ) , d y n o r p h i n and several e n k e p h a l i n s are found in the interior o f their precursors, fl-Endorphin and Met-
e n k e p h a l i n - A r g - P h e o c c u r at the e x t r e m e C O O H - t e r m i n a l e n d o f their parent m o l e c u l e s . T h e A and B c h a i n s o f relaxin are d e r i v e d from o p p o s i t e e n d s o f the s a m e p r e c u r s o r m o l e c u l e ; the m i d d l e , C region, is r e m o v e d d u r i n g processing. M o r e than one b i o l o g i c a l l y active peptide can be d e r i v e d from a s i n g l e p r e c u r s o r or polyprotein. A C T H , o~-melanotropin ( a M S H ) and f l - e n d o r p h i n c o m e from a common p r e c u r s o r m o l e c u l e ; several c o p i e s o f the e n k e p h a l i n s are d e r i v e d from one larger m o l e c u l e ; d y n o r p h i n and the n e o - e n d o r p h i n s share a s i n g l e parent m o l e c u l e . A t one t i m e it w a s a s s u m e d that o n l y one b i o l o g i c a l activity w o u l d c o m e from one p r e c u r s o r m o l e c u l e , but it has n o w b e c o m e c o m m o n practice to e x a m i n e e v e r y region o f a precursor for a p o s s i b l e b i o l o g i c a l role. M a n y of the k n o w n proteolytic c l e a v a g e s o f precursors o c c u r at pairs o f basic a m i n o acid residues ( L y s or A r g ) (Fig. 1 and T a b l e I). W h e n such a c l e a v a g e occurs, both basic residues are usually absent from the resultant peptide products (for e x a m p l e c a l c i t o n i n and c a l c i t o n i n C O O H - t e r m i n a l c l e a v a g e peptide ( C C P ) ; A C T H and the h i n g e peptide; A C T H and /3-1ipotropin; A V P and n e u r o p h y s i n II). H o w e v e r , there are i m p o r t a n t e x c e p t i o n s in w h i c h one basic residue r e m a i n s w i t h a product peptide; corticotropin-like i n t e r m e d i a t e lobe peptide ( C L I P ) , ",/3MSH and ~/tMSH are found w i t h a basic residue at their NH2t e r m i n u s ~3, s u g g e s t i n g that the initial proteolytic e v e n t in those cases w a s not at the C O O H - t e r m i n u s o f the pair o f basic
TABLE 1. Amino acid sequences around some known cleavage sites in precursors
A. Cleavage at a pair of basic amino acids, with the pair absent from the products Basics Pig gastrin - Pro - Ala- Set - His - His ~ Arg- Arg~ Gin- Leu- Val - Ala - Asp- Leu- Ala~ Lys - Lys~. Gin - Gly- Trp - Met- Asp- Phe - Gly .~Arg - Arg[ Set - Ala Bovine AVP-neurophysin I1 - Cys - Pro - Arg - Gly - Gly ~ Lys - Arg [ Ala - MetBovine ACTH/endorphin - Gly - Pro - Arg- Glu - Asp~ Lys - Arg~ Ser - Tyr - Phe- Pro - Leu- Glu - Phe~. Lys - Arg'r Glu- Leu- Ser - Pro - Pro - Lys - Asp~ Lys - Arg ~ Tyr - Gly Bovine enkephalins - Tyr - Gly - Gly - Phe - Met rLLys - Arg ~ Tyr - Gly - Tyr - Gly - Gly - Phe - Mettr Lys - Arg.~Asp- Ala - Thr - Lys - Glu - Leu - Gin .~Lys - Arg [ Tyr - Gly - Tyr - Gly - Gly - Phe - Leu ~ Lys - Arg .~Phe - Ala Pig neo-endorphin/dynorphin - Pro - Lys - Glu - Gin - Val .~Lys - Arg ~rTyr - Gly - Leu - Arg - Lys - Tyr - Pro ~ Lys - Arg 'r Ser - Ser - His - Glu- Asp- Leu- Tyr~ Lys - ArgOtTyr- Gly- Lys - Trp - Asp -Asn - Gin ~ Lys - Arg r Tyr - Gly B. Cleavage COOH-terminal to a single basic residue Cleavage Bovine AVP-neurophysin I1 - Phe - Pro - Arg- Arg- Val - Arg~ Ala - AsnCholecystokinin - His - Arg - lie - Ser - Asp- Arg.~Asp- Tyr Rat somatostatin - Arg - Leu - Gin - Leu. Gin - Arg ~ Ser - Ala C. Cleavage with chymotrypsimlikespecificity Cleavage Bovine AVP-neurophysin II - Ser - Asn- Ala - Thr - Leu- Leu ~Asp- Gly - Ser - Gly - Ala - Leu - Leu- Leu~ Arg- LeuRat relaxin - Val - Gly - Arg - Leu - Ala - Leu i Set - Gin ~'~ 1983, Elsevier Science Publishers B V . , Amsterdanl
Ref. 45
25 29
32
21
Ref.
25 36 16 Ref~ 25
0378 - 5912/83/$01.00
TINS -June 1983
230 tissues producing the same precursor carry out the same set of proteolytic cleavages, For example, the common precursor to dynorphin and the neo-endorphins contains three copies of the sequence -Tyr-Gly-GlyPhe-Leu-Arg-Basic-, yet the cells produc-
residues. A basic residue remains at the COOH-terminus of c~-neo-endorphin but is removed from /3-neo-endorphin (which ends in Pro) 43. Not all pairs of basic residues actually serve as cleavage sites. In addition, not all g~strin (pig) |+~.".'.1
RR
KK
RR
II
II
II
-I
vo
~
p ~pLRI+A
H
AVP-Ne~
il (bovino) ~;,((.;;~ l l +
ing dynorphin and a-neo-endorphin do not contain : detectable amounts of Leuenkephalin (Tyr-Gly-Gly-Phe-Leu)"~. The ratio of o~-neo-endorphin to /3neo-endorphin is much greater than 1 in most brain regions, but is about 1 in the
I I I+I I I
LI
p . . . . . . . . . . . . . . . . . . . . .
• + .......
I
+. . . . . .
+am
+,++.~+ +
:,++
i
II LS
RK
KK
C Region
I
RKKR H A Ch°in I
lI
A G T H / e n d o ~ (bovine) I
K K~K u I H
enkoptmPas (bovine) p+~/+/~+~+~+~
,
KRKK n Ii H fl,
I
I
:I
?I •
I+
:
I
I
ME.Rt~L
I
KR KR
ti,,l
,,
(pig)
~ [
m~+mm~ (rat) +s+<+:/>+~/A
I
""~
I
i
,
RRRR
!
[. ......+]
KR TR
It+,
i
~R -
KR cak~oP~ l u n e ~
KR
'
"
I
I
CGRP Signal _I -25
i 0
I
I 50
I
I , ,I I00 Number of Amino Acids
I 150
I
~ 200
i
i 250
F~, l,moomav+ pepnde+: ~ ofselecteaprecursorsand some oftheirpro~c=. Dam aredev+veafrom the~ nqevences:gaxWn +~A-V P-mmvol~ys+. ~ 4,? II~lLII+17 ll~, r e ~ I I ; ( i L - ' i ~ and C G R P ; e n k e ~ ~ neo-em~rp/r~/d+wtorpkin S l , 4 L 4 M I ; A C T H + ; ;II,]141~4klI+. The ~ p m of ~ W~e~ ~he~im ~ e ~ ~+ep r ~ e m i~n~: ~n~wn f ~ m ~j¢p~ w~rk~ ~se~igna~~ ~ ~ ~ ~ ~ ~w ~ ~ ~ ~ ~ ~ u : Amino ac~Isojknown cleavage~ av¢~ by O~ ~ IeP~rcode (R = ~ ; K = ly~mw.,.L = ~ ; D =~ ~ V = ~; G --glyc+m~A = ~ ; S = serine;Q = ~ ; F = phen~; T = 6:veonine);upward marlcvindicatec l e a ~ known to oor.sor,and downwa~I maml~lare cleavage sitesw~ch are not known to be =O~zed. For some molecvJes(for ~ ~ E and F, ACTH, ~ the .pwan+ ~ ~ +~esol cleavage which are not always cleaved. The s~es n m r k ~ for the C ~ n o f rela~n" and all o f C G R t n are ~ : Abbt~im+ons are: A VP = vasopressin; A C T H = corlicoc~pin; MSH = ~ i n ; C L I P = cortlcotropin-ltke intermediate lobe l~p¢ide; M E = m~h~nine.enkephal~ L E = leucine-enkephal~n; CCP = caicisonin COOH-~rn~al cleavage pel~de; CGRP = caicitonin gene-related peptide.
TINS - J u n e 1 983
hypothalamus and posterior pituitary43. ACTH remains largely intact in the anterior pituitary, yet it is quantitatively cleaved to produce c~MSH and CLIP in the intermediate pituitary, fl-Endorphin(l-31) remains intact in the anterior pituitary, while the -Lys-Lys- pair near the COOH-terminus of the peptide is slowly cleaved in the intermediate pituitary ~.4G. An interesting point comes from comparing the amino acid sequences at corresponding cleavage sites in precursors from different species; when a cleavage occurs at a pair of basic residues in the precursor from one species and the pair is not present in the precursor from a second species, the cleavage will not occur in that second species. In addition to the wellknown examples of the albumin and insulin mutants 9, there are examples within the ACTH/endorphin precursor: the pair of basic residues at the NH2-terminus of /3MSH and at the COOH-terminus of 7tMSH in the bovine precursor are absent from the rat precursor, and these smaller peptides are not formed in the rat pituitaryT, 1o. Not all proteolytic cleavages involved in prohormone processing occur at pairs of basic amino acids (Table I). Some cleavages occur immediately COOH-terminal to a single basic residue: in addition to the COOH-terminal end of neurophysin II, the NH2-terminal ends of somatostatin-28 and of cholecystokinin-8 fit into this general group of cleavages9"16. Other cleavages that do not occur at basic residues and might be considered chymotryptic in specificity include cleavages at the B-C chain junction in relaxin 3~, cleavages within the COOHterminal glycopeptide of the AVPneurophysin II precursor 25, and a cleavage within the C-peptide of insulinL Some care must be exercised in accepting the existence of chymotryptic (or non-tryptic) cleavages as genuine biosynthetic events, as some cleavages of this type (for example human /3MSH) have been shown to be artefacts of degradation after tissue homogenization TM. One very well studied example does not fit any of the above patterns - melittin24. Melittin is cleaved from promelittin by a succession of aminodipeptidase cleavages, eventually reducing the parent molecule to the product peptide. Usually the products produced by proteolytic processing of a precursor are stored and co-secreted in equimolar amounts. This result has been demonstrated for the AVP-neurophysin 25, insulin-C-peptide9, calcitonin-CCP2, pancreatic polypeptideicosapeptide (peptide IIIb) 3~, and ACTHendorphin systems of peptides. Even though some of the peptides produced seem to possess no biological activity (for exam-
231 pie insulin C-peptide), the ability to monitor their secretion and plasma levels may provide useful and sensitive measures of pathological over- and under-secretion of active peptide. Fig. 1 includes one example of what may prove to be a common and important posttranscriptional but pre-translationai modification; the pair of precursors diagrammed at the bottom of Fig. 1 (calcitonin and calcitonin gene-related peptide) appear to be derived from a single gene after alternate splicing at the mRNA levelz. Interestingly, the primary product of that mRNA rearrangement is tissue specific, in that thyroid C-cells produce almost exclusively mRNA for calcitonin, while hypothalamic neurons produce mRNA primarily coding for the calcitonin gene-related peptide. Proteolytic processing can either activate or inactivate a molecule. Virtually all of the known protein precursor molecules are biologically inactive, or at most they are active only at physiologically implausible concentrations. Thus proteolytic processing is required to produce a bioactive peptide. lntracellular proteolytic processing can also inactivate a peptide before secretion. Examples include the production of cholecystokinin-4, which lacks activity on the exocrine pancreas and gall bladde# 6, and the cleavage of ACTH to form aMSH and CLIP, which lack adrenal steroidogenic activity (however o~MSH has more melanotropic activity than ACTH). Extracellular proteolysis may often inactivate peptides, but the conversion of angiotensinogen to angiotensin I1 is a well-known case of activation22. The variety of smaller peptides that can be generated from a single precursor may be of use in targeting peptides for specific receptors. For example, somatostatin-28 is much more active than somatostatin-14 at inhibiting insulin secretion, yet the two forms of somatostatin are equipotent at inhibiting growth hormone and glucagon secretion 6. There are at least four distinct types of opiate receptors, which show unique patterns of selectivity for the several forms of /3-endorphin, dynorphin, neoendorphin, enkephalins and enkephalin precursors ~'s,26.4a. Several important points can be obtained from the above general observations and applied to studies of the enzymes responsible for the proteolytic processing occurring during biosynthesis. First, the subcellular site of proteolytic processing events is an important clue to the best place to search for the relevant enzymes. In particular, the proteolytic events occurring within about 30 min of precursor synthesis usually take place in the endoplasmic reticulum or Golgi apparatus, while any later cleavages occur
primarily in the secretory granules 9'~s. An organelle clearly not involved in normal biosynthesis is the lysosome: unfortunately, many studies of potential proteolytic processing enzymes do not include lysosomal marker enzyme assays, or else proceed in the presence of major lysosomal contamination. It is clear that tissues usually produce a rather homogeneous set of peptide products. Specifically, there are a great number of cleavages at pairs of basic residues, after which the basic residues are excised from the product peptides. Studies of the enzymes responsible for such cleavages should employ chemical characterizations of the products produced in vitro which can discern this type of difference in structure. Tissue-specific cleavage patterns are not infrequent and should be useful in the initial stages of enzyme characterization. Thus there are functionally distinct enzymes involved in cleavages at paired basic residues in ACTH/endorphin cells of the anterior pituitary (which produces ACTH) and the corresponding cells of the intermediate pituitary (which cleaves ACTH to create aMSH and CLIP). Similarly, there are functionally distinct enzymes in enkephalin-producing cells and in dynorphin/neo-endorphin-producing cells (recall Fig. 1)42'4a. With only a couple of physiologically important exceptions (for example when insulin or parathyroid hormone secretion is suppressed), there is remarkably, little intraeellular degradation of peptides. Peptides are frequently stored for days after production in the same secretory granules in which the precursors were efficiently cleaved to the product peptides. Therefore it is reasonable to expect an enzyme, which is functioning in a test-tube like the biosynthetic cleavage enzyme in intact cells, to halt after the correct cleavages are completed. This criterion has not commonly been applied in evaluating the biological relevance of potential proteolytic processing enzyme preparations. Sometimes the order of cleavages within a precursor and the rates at which those cleavages occur can be exploited. For example, in the processing of proACTH/endorphin there is a rather rigid order of cleavages. First, /3-1ipotropin is cleaved from the precursor, and on a much slower time-scale /3-endorphin is liberated from /3-1ipotropin; seldom does the /3-endorphin get cleaved directly from the common precursor. By contrast, the cleavages of proinsulin to form insulin and C-peptide seem to occur at about the same time, with several biosynthetic intermediates present in comparable amounts 9.
TINS -June 1983
232 TABLE II. Selected amidated peptides and their precursor proteins, The amino acid sequences around known amidation sites are shown. The residue that will he amidated is tmdm'lined; adjacent Gly residues are circled; adjacent basic amino acids are doubly underlined. Hinge peptide (human and rat) and N-terminal glycopeptide (bovine or pig) are fragments of pro-ACTH/endorphin (see Fig. 1). References: hinge peptide4,',*°;melittinu; calcitonin2; vasopressin~; aMSH~; gastfin45;N.terminal glycopeptide
Peptide
Asn Glu
VIP Hinge peptide
-Pro-Arg-G..~..l~@
Gin
Melitlin
-Arg-Gln-_~ar ~31~ -OH
Pro
Calcitonin TRH Vasopressin LHRH Corticotropin-releasing factor Secretin c~MSH Gaswin-relensingpeptide PHI PYY Neuropcptide Y Pancreatic poly~ptide Gastrin
-'Gly-Ala-~ ~9-Lys-Lys-Arg-Asp-
Gly Ala Val Met lie Tyr
Phe
Precursor ? -L~s-Ar~-Ser-Tyr-
~
- Pro-Arg-Gly. ~,Lys-Arg-Ala-Met? ? -Lys-Pro-Val- ~9-Lys-Lys-A~.Arg. ? ? ? ?
N-terminal glycopeptide
There are clearly a large number of precursors to bioactive peptides, and there must also be at least several distinct proteolytic processing activities involved in the biosynthesis of the smaller peptides. The proteolytic enzymes seem to function in the endoplasmic reticulum and Golgi apparatus as well as in secretory granules. There are several distinct sequence spocificities exhibited by biosynthetic proteolytic enzymes;
help to solve some of these problems. There have been too many studies of proteolytic processing enzymes to provide a satisfactory overview of these data in this brief article; interested readers are referred to some recent reviewss,*s,3s,4~. Thel:e clearly remains a tremendous amount to be learned about biosynthetic proteolytic processing enzymes.
-Met-Asp-Lt~e- ~
-Arg-Arl~Ser-Al&
-Asp-Arg-Phe- ~
-Arg-Arg-Asn-Gly-
Both endopeptidases and exo~ptidases must he utilized; but there is not yet any clear idea of how many differentenzymes are involved. It is not known whether each peptide system has its own unique enzymes, or whether several generic enzymes accomplish most of the known cleavages. The ability to incorporate genes coding for these pro-rnolecdes into other cell types and follow their expression may
n Many bioactive poptides have carboxyl terminal o¢-amide residues (Table II). In general, presence of the a-amide is critical for biological activity: for example, the potency of corticotropin-releasing factor is diminished at least 1 000-fold when Ala replaces Ala-NI-k4. and deamidated analogs of gastrin and cholecystokinin are totally inactiveSt A variety of amino acids are amidated, but current data suggest that amino acids with charged side chains are amidated less frequently. In promelittin. the Gly following melittin is the carboxyl terminus of the molecule and is not followed by a pair of basic residues. In other cases where data are available, the precursor proteins to amidated peptides all contain the amino acid sequence -X-Gly-Basio.Basic- where X is the residue that becomes amidated in the mature peptide and the basic residues can be Lys or Arg. This presumed amidation plus proteolytic cleavage recognition site has been seen so universally that precursor proteins containing it (based on nucleotide analysis)
TABLE IlL Acetylation of aMSH and/3-endorphin Peptide
otMSH
Effect of a-N-acetylation on bioactivity 0
0
0
0
0
II
I[
II
H
t~
Nl-h-CH-C.Peptide ~ Cl-h-C-NH-CH-C-Peptide -* CI.-h-C-NH-CH-C-Peptide
I
t
I
CH2
CH2
CH2
OH
OH
O
Greatly increases skin-darkening potency
I
C=O
I
ACTH(I- 13)Nl-h
~-endorphin
a-N-acetyl- ACTH( 1-13)Nl-h
0
0
0
II
II
II
c¢-N,O-thacetyl-ACTH(l- 13)Nl-h
Nl-h.CH-C.-Peptide --~ Cl-h-C-NH-CH.C- Peptide
5 CI-G
© L
OH /3-endorphin(I-31)
CH,
© ~
Oil a- N-acetyl-~-endorphin( 1,3 i )
Greatly decreases opiate potency
233
T I N S - J u n e 1983
are often assumed to lead to a product peptide ending in -X-NFh (for example yl MSH, hinge peptide, calcitonin generelated peptide)2,4.t3. While such a sequence may be necessary for amidation to occur, it is not sufficient: the pro-ACTH/endorphin molecule contains the sequence - Val-Glys-Lys-Lys-Arg-Argwhich is converted into the VaI-NH2 of txMSH in intermediate pituitary cells but remains intact as the mid-region of ACTH(1-39) in anterior pituitary corticotropes. Tatemoto and MutP 9 noted that peptides with carboxyl terminal a-amides occur primarily among neural and hormonal peptides. Therefore they developed a chemical detection system for peptides containing carboxyl terminal a-amides. In this way they identified several previously unknown bioactive peptides. The same 27-residue secretin/glucagon/VIP/GIP-like peptide terminating in Ile-NH2 was found in extracts of pig intestine and brain. Two similar but distinct 36-residue pancreatic polypeptide-like peptides both terminating in Tyr-NH~ were found; one was localized. to brain and the other to intestine. Bradbury et al.5 recently succeeded in characterizing an amidating activity in extracts of pig pituitary secretory granules. This enzyme converts synthetic tripeptides ending in the sequence -X-GIy into the corresponding des-Gly peptide amide; X can be a neutral amino acid like Val, Phe or Gly, but not a charged amino acid like Asp or Lys. The nitrogen of the peptide amide comes from the a-amino group of Gly, and glyoxylate is released as a reaction product (Fig. 2). The substrate specificity of the pituitary enzyme suggests that the same enzyme could amidate aMSH, gastrin, cholecystokinin, oxytocin and vasopressin. The amidating ability of peptide-producing cells may show interesting regulatory properties: cultured dog pseudoislets continue to produce pro-pancreatic polypeptide and convert it into a pancreatic polypeptidesized molecule, but amidation no longer occurs after a period of time in cultureW"~7; similarly, although cultured rat intermediate pituitary cells produce aMSH-sized peptides, the peptides are no longer amidated and terminate with -Gly~4-OH.
Aeetylation Co-translational NH=-terminal acetylation of proteins is common, but its physiological role is not known. The posttranslational acetylation of histones on the e-Nl-h group of Lys has been extensively studied and may play a role in gene regulation. Among neural and hormonal peptides, post-translational Nl-h-terminal acetylation is an important modification for aMSH and
HOHHO
HO
I II I I II Peptide-C-C-N-C-C-OH I I R H residue to be
I II + Y
glycine
HO
I il
= Peptide-C-C-N=C-C-OH I R
+ YH2
unstable imine
amidated
HO
HO HO O0 I II I ,I li II Peptide-C-C-N=C-C-OH + H20------'~Peptide-C-C-NH2 + H-C-C-OH I I
I II
R
unstable imine
R
amidated peptide
glyoxylate
Fig. 2. Proposed mechanism for amidation. (Adapted from the reaction mechanism proposed by Bradbury et al. 5 The necessary oxidizing agent Y was not specified. )
/3-endorphin. In addition, about 5% of rat growth hormone=7 and 25% of Leuenkephalin in the neural lobe of the rat pituitary are NH2-terminally acetylated; interestingly, dynorphin, a 17-residue opioid peptide found in the hypothalamus and neural lobe of the pituitary, contains the Leu-enkephalin sequence at its Nl-hterminus but is not acetylated38. Given the dramatic effects of NHa-terminal acetylation on the bioactivity of aMSH, fl-endorphin and Leu-enkephalin, it will be of interest to look for this modification in other systems. Acetylation is one of the tissue-specific post-translational modifications involved in the processing of pro-ACTH/endorphin and occurs in the intermediate but not in the anterior pituitary (Table III). Acetylation of the NI-I~-terminus of ctMSH is required for potent melanocyte-stimulating activity while acetylation of/3-endorphin abolishes opiate activity; a single post-translational modification has evolved that activates one peptide (aMSH) while inactivating another (fl-endorphin). In the intermediate pituitary of several species, aMSH exists in three acetylation states: desacetyl-aMSH or ACTH(1-13) NI-h, aMSH or a-N-acetylACTH(1-13)NI-h, and a-N,O-diacetylACTH(1-13)NI-h7,15,sa. Pulse-chase labelling experiments have recently shown that the pathway for the acetylation of ACTHrelated molecules in the rat intermediate pituitary begins with desacetyl-aMSHsized molecules which are precursors to mono- and di-acetylated peptides15. There is some controversy regarding the acetylation of ACTH- and /3endorphin-related molecules in the brainx'2s.ss. Zakarian and Smyth*~ reported
finding primarily/3-endorphin(1-31) in the rat hypothalamus (the region containing fl-endorphin-producing nerve cell bodies) and acetylated forms in the hippocampus, colliculae and brain stem (primarily fiber terminal areas). Other studies have suggested the presence of only non-acetylated forms of fl-endorphin and aMSH in rat brain14. Non-specific acetylating activity has been found in homogenates of many tissues (for example pituitary, liver, lung, brain)3a.44; the enzyme activity responsible for the co-translational acetylation of proteins (probably associated with the rough endoplasmic reticulum of all tissues) is able to acetylate ACTH- and /3endorphin-related molecules in vitro. Biosynthetic labelling experiments coupled with subcellular fractionation of rat intermediate pituitary cells have shown that fl-endorphin(l-31 ), ACTH( 1-13)NI-h, and aMSH become NH~terminally acetylated when associated with secretory granules15. The secretory granules of the intermediate pituitary but not those of the anterior pituitary contain peptide acetyltransferase activity believed to be responsible for the acetylation of ACTH- and /3-endorphinrelated molecules in vivo. The secretorygranule-associated acetyltransferase from bovine intermediate pituitary acetylates ACTH(1-13)NI-h, aMSH and fl-endorphin(1-31). The acetylated forms of /3-endorphin inhibit the acetylation of both /3-endorphin and ACTH-related molecules and steady-state kinetic experiments have shown that this inhibition is competitivelY; this suggests that the same enzyme acetylutes both ACTH- and fl-endorphin-related peptides.
234
TINS - June 1983
TABLE IV. Sulfatedpeptides Phosphorylation The description of phosphorylated Peptide Sequencearoundsulfationsite Effect of sutfationon bioactivity ACTH and CLIP in rat pituitary marked the first time that naturally occurring small SOsH bioactive peptides were shown to be phost phorylateda.L In corticotropes of the rat Gastrin -Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Nl'h No effecton stimulationof gastric acid secretion anterior pituitary approximately half of the ACTH molecules are phosphorylated at SO,H SeP t , while in the rat intermediate pituitary Cholecystokinin . Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-Nl-h Essentialfor actionon gall bladder approximately two-thirds of the CLIP and pancreas molecules are phosphorylated. The degree SO3H of phosphorylation of ACTH and CLIP is 1 pyroGlu-Glrv Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-Nl-h species specific: mouse pituitary ACTH Caerulein and CLIP are phosphorylated 3-fold less S(hH I than the rat molecules; bovine pituitary Leu-enkephalin Tyr-Gly-Gly-Phe-Leu Inactivates optate bioactivity ACTH and CLIP are not phosphorylated; and about one-third of human ACTH is phosphorylateda, 12. Phosphorylation is a protein kinase- Sulfafian terminal N- acetylgalactosamine and mediated process which can be divided into Gastrin, cholecystokinin (CCK) and the N-acetylghtcosamine residues of the general mechanisms based on substrate amphibian peptid¢ ¢a~rdein represent a asparagine-linked oligosaceharide*'. This specificity. Cyclic nucleotide-dependent family of peptides derived from a common type of sulfation has been observed extenand calcium-dependent protein kinases ancestral peptide. The COOH-terminal sively in proteoglycan synthesiss° and phosphorylate serine residues located to the pentapcptid¢ of these hormones is identical recent evidence indicates that this mechanCOOH-terminal side of groupings of basic and this regionrepresents the active site of ism may be important for other pituitary amino acids 23, while the physiological both gastrin and CCK. In addition, the glycoproteins. Bovine thyrotropin and casein kinases phosphorylate Ser or Thr COOH-terminal region is also the site of human hiteinizing hormone also appear to residues where the sequence 1S sulfation for then~ peptides (Table IV). be sulfated like bovine luteiuizing hor-Ser(Thr)-X-Acidic-3t Phosphorytation of As reviewed by Rehfetd's, CCK is mone, while human chorionic gonadotrorat ACTH and CLIP occurs at the sequence synthesized by cells of the intestine and pin is not sulfated. Sulfation does not affect -Seraa-Ala3'-Glu3a-; in bovine ACTH, brain as part of a precursor protein consist- the ability of these hormones to bind to Glu 3a is replaced by Gin s3 and phosphoryla- ing of approximately 130 amino acids. Tim target tissue, however it is speculated that tion does not occur. This specificity thus precursor ,s successively cleaved to yield sulfated terminal sugars may function like resembles that of the physiological casein CCK-39, CL-'K-33, CCK-12, CCK,8 and negatively charged sialic acid residues to kinases. The rat pro-ACTH/endorphin CCK-4. All of the forms of CCK except enhance the half-lives of these hormones. molecule has potential phosphorylation CCK-4 arc sulfated and this modification is Rat pro-ACTH/endorphin may be sulfated sites in the Nl-h-terminal glycoprotein essential for stimulating gall.bL_a,~_~ empin a similar fashion *°. (Se# 8) and in o~MSH (Sera of ctMSH); tying and secretion of pancreatic enzymes. phosphorylatcd c~MSH has not been obGastrin is synthesized by cells of the A c k n o ~ n l s We thank out many colleaAgueswho provided served and the NH~.terminal glycoprotein stomach as part of a 105-amino-acid prepreimnts of their work and apologize for our inais phosphorylated to a much lesser extent cursor protein which is cleaved in a series of bility to cover all of the vast amount of relevant than ACTH or CLIP TM. Thus the sequence. steps to gastrin-34 and then to gastrin- 17. In material in this review. Research was supported Ser-X-Acidic- appears to be just part of the contrast to CCK, only approximately half by NIH grants AM-18929, AM-19859, signal for initiating phosphorylation. of the g a s ~ r e l a t e d forms are sulfated and NS-06648 and NS-06363, and a grant from the Studies of biosynthesis indicate that sulfation has no effect on the ability of McKnight Foundation, phosphorylation occurs shortly after syn- gastrin- 17 to stimulate gastric acid secrethesis of p r o - A C r H / ~ , l~-sama~y tion in mammals. It is not clear when during Reading list 1 Akil, H.. Shiomi.H.. Walker.J. M. and Watson. in the endoplasmic reticulum or Golgi at biosynthesis gastrin or its precursor is S. (1982)Adv. Biochem. PsychopharmacoL roughly the time that glycosylation of the sulfated. 33, 61-67 precursor is occurring. The two processes In addition to the gastrin-CCK related 2 Amara. S. G.. Jonas. V.. Rosenfeld. M. G., Ong, probably occur sequentially (glycosylation, peptides, Len-enkephalin with an OE. S. and Evans,R. M. (1982)Nature (LondonJ 298. 240-244 then phosphorylation) and neither seems to sulfated tymsyt residue has been isolated 3 Bennett, H. P. J., Browne, C. A. and Solomon,S. greatly affect the biological activity of from brain*'. Sulfated Lcu-enkephalin (1981) Proc. Nail Acad. Sci. USA 78. ACTH s, t,. represents 10-20% of the L e w e n k ~ 471"3-4717 Many other neural hormonal polypep- isolated from strintam and is biologically 4 Boileau, G,, Laraviere. N., HsL K.-L., Seidah. tides have potential sites for this type of inactive. At present it is not known wl,~hex N. G. and Chretien,M. (1982)B/ochem/stry21. phosphorylation. For example, numbering any of the other many enkephalin-related 5341-5346 with the first residue of the pro-molecule as peptides are sulfated. 5 Bradbury, A. F.. Finni¢, M. D. A. and Smyth, D. G. (1982)Nam.~ (London) 298,686-.688 No. 1, phosphorylation could occur at Very little is known about the sulfoffans6 Brown, M.. Rivier. J. and Vale. W. (1981) Ser ~ of pig pro-gastrin~; at Se# °+~t femse activities that must be involved in Endocrinology t08.2391-2393 and ThP"s of rat pro-calcitonin (assuming a linking sulfate to these specific tyrosyl 7 Browne,C. A., BenncR.H. P. J. and Solomon,S. 22-amino-acid signal peptide)'; at residues. (1982)Aria/. Biochem. 124,201-208 Sera''lS'''a and Thr s¢'*~s of bovine proBovine luteinizing hormone occurs in 8 CocbcR,A. D., Paterson.S. J.. MeKnight,A. T.. enkephalin in AS'; at S e t as'Ts't~'*6s'~tt of sulfated and non, sulfated forms, but in Magnan,J. and Kos~flitz, H. W. (1982)Nature pig pro-enkephalin B tt. (London) 299, 79-81 this case the sulfate is linked to the
TINS -June 1983 9 Docherty, K. and Steiner, D. F. (1982) Annu. Rev. Physiol. 44, 625--638 10 Drouin, J. and Goodman, H. M. (1980) Nature (London) 288, 610-613 11 Eipper, B. A. and Mains, R. E. (1981)J. Biol. Chem. 256, 5689-5695 12 Eipper, B. A. and Mains, R. E. (1982) J. Biol. Chem. 257, 4997-4915 13 Esch, F. S., Shibasaki, T., Bohlen, P., Wehrenberg, W. B. and Ling, N. C. (1981) in Peptides, (Rich, D. H. and Gross, E., eds), pp. 485-488, Pierce Chemical Co., Rockford, IL 14 Evans, C. J., Lorenz, R., Weber, E. and Barchas, J. D. (1982)Biochem. Biophys. Res. Commun. 106, 910-919 15 Glembotski, C. C. (1982) J. Biol. Chem. 257, 10493-10500 16 Goodman, R. H., Jacobs, J. W., Dee, P. C. and Habener, J. I?. (1982)J. Biol. Chem. 257, 1156-1159
17 Herbert, E.. Bimberg, N., Lissitsky, J. C., Civelli, O. and Uhler, M. (1981)Neuroscience Newsletter 12, 16-27 18 Hope, J. and Lowry, P. J. (1981)Front. Horm. Res. 8, 44-61 19 Hortin, G., Natowicz, M., Pierce, J., Baenziger, J., Parsons, T. and Boime, 1. (1981) Proc. Natl Acad. Sci. USA 78, 7468-7472 20 Hoshina, H., Hortin, G. and Boime, 1. (1982) Science 217, 63434 21 Kakidani, H., Furutani, Y.,Takahashi, H., Noda, M.. Morimoto, Y., Hirose, T., Asai, M., lnayama, S., Nakanishi, S. and Numa, S. (1982) Nature (London) 298,245-249 22 Kotchen~ T. A. and Guthrie, G. P. (1980) Endocr. Rev. 1, 78-99 23 Krebs, E. G. and Beavo, J. A. (1979) Annu. Rev. Biochem. 48,923-959
235 24 Kreil, G., Mollay, C. Kaschnitz, R., Haiml, L. and Vilas, V. (1980) Ann. N Y Acad. Sci. 343, 338-345 25 Land, H., Schutz, G., Schmale, H. and Richter, D. (1982) Nature (London) 295,299-303 26 Lewis, R. V. and Stem, A. S. (1983)Annu. Rev. Pharmacol. Toxicol. 23,353-372 27 Lewis, U. J., Singh, R. N. P., Titirler, G. F., Sigel, M. B., Vanderlaan, E. F. and Vanderlaan, W. P. (1980) Recent Prog. Horm. Res. 36, 477-508 28 Liotta, A. S., Yamaguchi, H. and Krieger, D. T. (1981)J. Neurosci. 1,585-595 29 Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N. and Nurna, S. (1979) Nature (London) 278,423-427 30 Neufeld, E. F. and Ashwell, G. (1980) in Biochemistry o f Glycoproteins and Proteoglycans, pp. 241-266, Plenum, New York 31 Niall, H., Hudson, P., Haley, J., Cronk, M. and Shine, J. (1982)Ann. NYAcad. Sci. 380, 13-21 32 Nod,a, M., Fumtani, Y., Takahashi, H., Toyosato, M , Hirose, T., lnayama, S., Nakanishi, S. and Numa, S. (1982)Nature (London) 295,202-206 33 O'Donohue, T. L. and Dorsa, D. M. (1982) Peptides 3,353-395 34 Paquette, T. L., Gingerich, R. and Scharp, D. (1981) Biochemistry 20, 7403-7408 35 Pinna, L. A., Meggio, F. and Bonella-Deana, A. (1980) in Protein Phosphorylation and Bioregulation (Thomas, G., Podesta, E. J. and Gordon, J., eds), pp. 8-16, S. Karger, New York 36 Rehfeld, J. F. (1981) Am. J. Physiol. 240, 6255-6266 37 Schwartz, T. W. and'l'ager, H. S. (1981)Nature (London) 294, 589-591 38 Seizinger, B. R., Hollt, V. and Herz, A. (1982)J. Neurochem. 39, 143-148
39 Tatemoto, K., Carlquist, M. and Mutt, V. (1982) Nature (London) 296, 659--660 40 Unsworth, C. D. and Hughes, J. (1982) Nature (London) 295,519--522 41 Vale, W., Spiess, J., Rivier, C. and Rivier, J. (1981)Science 213, 1394--1397 42 Watson, S. J. and Akil, H. (1982)Adv. Biochem. Psychopharmacol. 33, 33--42 43 Weber, E., Evans, C. J., Chang, J. K. and Barchas, J. D. (1982) Biochem. Biophys. Res. Commun. 108, 81-88 44 Woodford, T. A. and Dixon, J. E. (1979)J. Biol. Chem. 254, 4993--4999 45 Yoo, O. J., Powel|, C. T. and Agarwal, K. L. (1982) Proc. Natl Acad. Sci. USA 79, 1049-1053 46 Zakarian, S. and Smyth, D. G. (1982)Biochem. J. 202, 561-571
Richard E. Mains, Betty A. Eipper, Christopher C. Glembotski and Robert M. Dores were, at the time o f writing this review, at the Department of Physiology, C-240, University o f Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262, USA. Richard E. Mains and Betty A. Eipper are now at the Department o f Neuroscience, Johns Hopkins University School o f Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. Christopher C. Glembotski is now at the Department o f Pharrnacology, University o f Pennsylvania School o f Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104, USA. Robert M. Dores is now at the Mental Health Research Institute, University o f Michigan School of Medicine, Ann Arbor, M148109, USA.
229
Strategies for the biosynthesis of bioactive peptides Richard E. Mains, Betty A. Eipper, Christopher C. Glembotski and Robert M. Dores Neuropeptides and small peptide hormones are usually synthesized as part o f large, inactive precursor proteins. Although the sequences o f many o f these pro-molecules are now known, much remains to be learned about the post-translational processing events that must occur to generate the bioactive products. W i t h the recent d e v e l o p m e n t o f rapid D N A s e q u e n c i n g techniques, o u r k n o w l e d g e o f precursors to s m a l l b i o a c t i v e peptides has g r o w n t r e m e n d o u s l y . For m a n y peptides w e k n o w the s e q u e n c e o f the pre-prom o l e c u l e and the structure o f the final b i o a c t i v e peptide product, but w e h a v e v e r y little k n o w l e d g e o f the i n t e r v e n i n g b i o s y n t h e t i c events. In this r e v i e w w e h a v e c h o s e n to d i s c u s s the f o l l o w i n g s o m e w h a t arbitrary subset o f the m a n y m o d i f i c a t i o n s k n o w n to o c c u r in n e u r o p e p t i d e and peptide h o r m o n e b i o s y n t h e s i s : proteolysis, amidation, acetylation, p h o s p h o r y l a t i o n and sulfation. O t h e r r e v i e w s can b e c o n s u l t e d for discussions of two important modifications that are not c o n s i d e r e d here: the function and r e m o v a l o f s i g n a l peptides and the addition and m a t u r a t i o n o f a s p a r a g i n e - l i n k e d o l i g o s a c c h a r i d e chains; in addition, w e h a v e not a t t e m p t e d to r e v i e w the vast literature on purification and characterization o f proteolytic p r o c e s s i n g e n z y m e s . W e h o p e to c o n v e y to TINS readers s o m e o f our e x c i t e m e n t about h o w m u c h r e m a i n s to be d i s c o v e r e d about the b i o s y n t h e s i s o f bioactive p e p t i d e s * . Precursors and products S i n c e protein precursors are a l w a y s bigger than their product peptides, proteases play a k e y role in the b i o s y n t h e s i s o f all the
p e p t i d e s to be d i s c u s s e d . S e l e c t e d e x a m ples o f protein precursors and s o m e o f their p r o d u c t s are s h o w n in Fig. 1. Our initial d i s c u s s i o n w i l l be c o n f i n e d to the o v e r a l l d e s i g n o f p r e c u r s o r s and their product peptides. B y o b s e r v i n g i m p o r t a n t patterns in the structural r e l a t i o n s h i p s o f p r e c u r s o r s to their products, w e hope to r e a c h s o m e con* Due to the restriction on the number of references allowed in this review, we have cited only the work most relevant to our limited discussion; there are many other studies that have been key in elucidating the mechanisms of bioactive peptide biosynthesis. In general, we have cited only one or two papers from a research group, literature survey completed August 1982.
c l u s i o n s r e g a r d i n g the proteolytic processing that results in the f o r m a t i o n o f the p r o d u c t peptides. First, the size o f the p r e c u r s o r protein is not s i m p l y c o r r e l a t e d w i t h the size o f the b i o l o g i c a l l y a c t i v e product peptide. For example, methionine-enkephalin (ME) and" l e u c i n e - e n k e p h a l i n ( L E ) are e a c h o n l y five a m i n o acids in length, yet they are d e r i v e d from one o f the b i g g e s t m o l e c u l e s s h o w n in Fig. 1. The b i o l o g i c a l l y active p r o d u c t peptide can o c c u r a n y w h e r e w i t h i n the structure o f its precursor. A r g i n i n e v a s o p r e s s i n ( A V P ) o c c u r s at the e x t r e m e N H ~ t e r m i n u s o f the p r o h o r m o n e , next to the s i g n a l peptide. Gastrin, c a l c i t o n i n , a d r e n o c o r t i c o t r o p i c h o r m o n e ( A C T H ) , d y n o r p h i n and several e n k e p h a l i n s are found in the interior o f their precursors, fl-Endorphin and Met-
e n k e p h a l i n - A r g - P h e o c c u r at the e x t r e m e C O O H - t e r m i n a l e n d o f their parent m o l e c u l e s . T h e A and B c h a i n s o f relaxin are d e r i v e d from o p p o s i t e e n d s o f the s a m e p r e c u r s o r m o l e c u l e ; the m i d d l e , C region, is r e m o v e d d u r i n g processing. M o r e than one b i o l o g i c a l l y active peptide can be d e r i v e d from a s i n g l e p r e c u r s o r or polyprotein. A C T H , o~-melanotropin ( a M S H ) and f l - e n d o r p h i n c o m e from a common p r e c u r s o r m o l e c u l e ; several c o p i e s o f the e n k e p h a l i n s are d e r i v e d from one larger m o l e c u l e ; d y n o r p h i n and the n e o - e n d o r p h i n s share a s i n g l e parent m o l e c u l e . A t one t i m e it w a s a s s u m e d that o n l y one b i o l o g i c a l activity w o u l d c o m e from one p r e c u r s o r m o l e c u l e , but it has n o w b e c o m e c o m m o n practice to e x a m i n e e v e r y region o f a precursor for a p o s s i b l e b i o l o g i c a l role. M a n y of the k n o w n proteolytic c l e a v a g e s o f precursors o c c u r at pairs o f basic a m i n o acid residues ( L y s or A r g ) (Fig. 1 and T a b l e I). W h e n such a c l e a v a g e occurs, both basic residues are usually absent from the resultant peptide products (for e x a m p l e c a l c i t o n i n and c a l c i t o n i n C O O H - t e r m i n a l c l e a v a g e peptide ( C C P ) ; A C T H and the h i n g e peptide; A C T H and /3-1ipotropin; A V P and n e u r o p h y s i n II). H o w e v e r , there are i m p o r t a n t e x c e p t i o n s in w h i c h one basic residue r e m a i n s w i t h a product peptide; corticotropin-like i n t e r m e d i a t e lobe peptide ( C L I P ) , ",/3MSH and ~/tMSH are found w i t h a basic residue at their NH2t e r m i n u s ~3, s u g g e s t i n g that the initial proteolytic e v e n t in those cases w a s not at the C O O H - t e r m i n u s o f the pair o f basic
TABLE 1. Amino acid sequences around some known cleavage sites in precursors
A. Cleavage at a pair of basic amino acids, with the pair absent from the products Basics Pig gastrin - Pro - Ala- Set - His - His ~ Arg- Arg~ Gin- Leu- Val - Ala - Asp- Leu- Ala~ Lys - Lys~. Gin - Gly- Trp - Met- Asp- Phe - Gly .~Arg - Arg[ Set - Ala Bovine AVP-neurophysin I1 - Cys - Pro - Arg - Gly - Gly ~ Lys - Arg [ Ala - MetBovine ACTH/endorphin - Gly - Pro - Arg- Glu - Asp~ Lys - Arg~ Ser - Tyr - Phe- Pro - Leu- Glu - Phe~. Lys - Arg'r Glu- Leu- Ser - Pro - Pro - Lys - Asp~ Lys - Arg ~ Tyr - Gly Bovine enkephalins - Tyr - Gly - Gly - Phe - Met rLLys - Arg ~ Tyr - Gly - Tyr - Gly - Gly - Phe - Mettr Lys - Arg.~Asp- Ala - Thr - Lys - Glu - Leu - Gin .~Lys - Arg [ Tyr - Gly - Tyr - Gly - Gly - Phe - Leu ~ Lys - Arg .~Phe - Ala Pig neo-endorphin/dynorphin - Pro - Lys - Glu - Gin - Val .~Lys - Arg ~rTyr - Gly - Leu - Arg - Lys - Tyr - Pro ~ Lys - Arg 'r Ser - Ser - His - Glu- Asp- Leu- Tyr~ Lys - ArgOtTyr- Gly- Lys - Trp - Asp -Asn - Gin ~ Lys - Arg r Tyr - Gly B. Cleavage COOH-terminal to a single basic residue Cleavage Bovine AVP-neurophysin I1 - Phe - Pro - Arg- Arg- Val - Arg~ Ala - AsnCholecystokinin - His - Arg - lie - Ser - Asp- Arg.~Asp- Tyr Rat somatostatin - Arg - Leu - Gin - Leu. Gin - Arg ~ Ser - Ala C. Cleavage with chymotrypsimlikespecificity Cleavage Bovine AVP-neurophysin II - Ser - Asn- Ala - Thr - Leu- Leu ~Asp- Gly - Ser - Gly - Ala - Leu - Leu- Leu~ Arg- LeuRat relaxin - Val - Gly - Arg - Leu - Ala - Leu i Set - Gin ~'~ 1983, Elsevier Science Publishers B V . , Amsterdanl
Ref. 45
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TINS -June 1983
230 tissues producing the same precursor carry out the same set of proteolytic cleavages, For example, the common precursor to dynorphin and the neo-endorphins contains three copies of the sequence -Tyr-Gly-GlyPhe-Leu-Arg-Basic-, yet the cells produc-
residues. A basic residue remains at the COOH-terminus of c~-neo-endorphin but is removed from /3-neo-endorphin (which ends in Pro) 43. Not all pairs of basic residues actually serve as cleavage sites. In addition, not all g~strin (pig) |+~.".'.1
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i
i 250
F~, l,moomav+ pepnde+: ~ ofselecteaprecursorsand some oftheirpro~c=. Dam aredev+veafrom the~ nqevences:gaxWn +~A-V P-mmvol~ys+. ~ 4,? II~lLII+17 ll~, r e ~ I I ; ( i L - ' i ~ and C G R P ; e n k e ~ ~ neo-em~rp/r~/d+wtorpkin S l , 4 L 4 M I ; A C T H + ; ;II,]141~4klI+. The ~ p m of ~ W~e~ ~he~im ~ e ~ ~+ep r ~ e m i~n~: ~n~wn f ~ m ~j¢p~ w~rk~ ~se~igna~~ ~ ~ ~ ~ ~ ~w ~ ~ ~ ~ ~ ~ u : Amino ac~Isojknown cleavage~ av¢~ by O~ ~ IeP~rcode (R = ~ ; K = ly~mw.,.L = ~ ; D =~ ~ V = ~; G --glyc+m~A = ~ ; S = serine;Q = ~ ; F = phen~; T = 6:veonine);upward marlcvindicatec l e a ~ known to oor.sor,and downwa~I maml~lare cleavage sitesw~ch are not known to be =O~zed. For some molecvJes(for ~ ~ E and F, ACTH, ~ the .pwan+ ~ ~ +~esol cleavage which are not always cleaved. The s~es n m r k ~ for the C ~ n o f rela~n" and all o f C G R t n are ~ : Abbt~im+ons are: A VP = vasopressin; A C T H = corlicoc~pin; MSH = ~ i n ; C L I P = cortlcotropin-ltke intermediate lobe l~p¢ide; M E = m~h~nine.enkephal~ L E = leucine-enkephal~n; CCP = caicisonin COOH-~rn~al cleavage pel~de; CGRP = caicitonin gene-related peptide.
TINS - J u n e 1 983
hypothalamus and posterior pituitary43. ACTH remains largely intact in the anterior pituitary, yet it is quantitatively cleaved to produce c~MSH and CLIP in the intermediate pituitary, fl-Endorphin(l-31) remains intact in the anterior pituitary, while the -Lys-Lys- pair near the COOH-terminus of the peptide is slowly cleaved in the intermediate pituitary ~.4G. An interesting point comes from comparing the amino acid sequences at corresponding cleavage sites in precursors from different species; when a cleavage occurs at a pair of basic residues in the precursor from one species and the pair is not present in the precursor from a second species, the cleavage will not occur in that second species. In addition to the wellknown examples of the albumin and insulin mutants 9, there are examples within the ACTH/endorphin precursor: the pair of basic residues at the NH2-terminus of /3MSH and at the COOH-terminus of 7tMSH in the bovine precursor are absent from the rat precursor, and these smaller peptides are not formed in the rat pituitaryT, 1o. Not all proteolytic cleavages involved in prohormone processing occur at pairs of basic amino acids (Table I). Some cleavages occur immediately COOH-terminal to a single basic residue: in addition to the COOH-terminal end of neurophysin II, the NH2-terminal ends of somatostatin-28 and of cholecystokinin-8 fit into this general group of cleavages9"16. Other cleavages that do not occur at basic residues and might be considered chymotryptic in specificity include cleavages at the B-C chain junction in relaxin 3~, cleavages within the COOHterminal glycopeptide of the AVPneurophysin II precursor 25, and a cleavage within the C-peptide of insulinL Some care must be exercised in accepting the existence of chymotryptic (or non-tryptic) cleavages as genuine biosynthetic events, as some cleavages of this type (for example human /3MSH) have been shown to be artefacts of degradation after tissue homogenization TM. One very well studied example does not fit any of the above patterns - melittin24. Melittin is cleaved from promelittin by a succession of aminodipeptidase cleavages, eventually reducing the parent molecule to the product peptide. Usually the products produced by proteolytic processing of a precursor are stored and co-secreted in equimolar amounts. This result has been demonstrated for the AVP-neurophysin 25, insulin-C-peptide9, calcitonin-CCP2, pancreatic polypeptideicosapeptide (peptide IIIb) 3~, and ACTHendorphin systems of peptides. Even though some of the peptides produced seem to possess no biological activity (for exam-
231 pie insulin C-peptide), the ability to monitor their secretion and plasma levels may provide useful and sensitive measures of pathological over- and under-secretion of active peptide. Fig. 1 includes one example of what may prove to be a common and important posttranscriptional but pre-translationai modification; the pair of precursors diagrammed at the bottom of Fig. 1 (calcitonin and calcitonin gene-related peptide) appear to be derived from a single gene after alternate splicing at the mRNA levelz. Interestingly, the primary product of that mRNA rearrangement is tissue specific, in that thyroid C-cells produce almost exclusively mRNA for calcitonin, while hypothalamic neurons produce mRNA primarily coding for the calcitonin gene-related peptide. Proteolytic processing can either activate or inactivate a molecule. Virtually all of the known protein precursor molecules are biologically inactive, or at most they are active only at physiologically implausible concentrations. Thus proteolytic processing is required to produce a bioactive peptide. lntracellular proteolytic processing can also inactivate a peptide before secretion. Examples include the production of cholecystokinin-4, which lacks activity on the exocrine pancreas and gall bladde# 6, and the cleavage of ACTH to form aMSH and CLIP, which lack adrenal steroidogenic activity (however o~MSH has more melanotropic activity than ACTH). Extracellular proteolysis may often inactivate peptides, but the conversion of angiotensinogen to angiotensin I1 is a well-known case of activation22. The variety of smaller peptides that can be generated from a single precursor may be of use in targeting peptides for specific receptors. For example, somatostatin-28 is much more active than somatostatin-14 at inhibiting insulin secretion, yet the two forms of somatostatin are equipotent at inhibiting growth hormone and glucagon secretion 6. There are at least four distinct types of opiate receptors, which show unique patterns of selectivity for the several forms of /3-endorphin, dynorphin, neoendorphin, enkephalins and enkephalin precursors ~'s,26.4a. Several important points can be obtained from the above general observations and applied to studies of the enzymes responsible for the proteolytic processing occurring during biosynthesis. First, the subcellular site of proteolytic processing events is an important clue to the best place to search for the relevant enzymes. In particular, the proteolytic events occurring within about 30 min of precursor synthesis usually take place in the endoplasmic reticulum or Golgi apparatus, while any later cleavages occur
primarily in the secretory granules 9'~s. An organelle clearly not involved in normal biosynthesis is the lysosome: unfortunately, many studies of potential proteolytic processing enzymes do not include lysosomal marker enzyme assays, or else proceed in the presence of major lysosomal contamination. It is clear that tissues usually produce a rather homogeneous set of peptide products. Specifically, there are a great number of cleavages at pairs of basic residues, after which the basic residues are excised from the product peptides. Studies of the enzymes responsible for such cleavages should employ chemical characterizations of the products produced in vitro which can discern this type of difference in structure. Tissue-specific cleavage patterns are not infrequent and should be useful in the initial stages of enzyme characterization. Thus there are functionally distinct enzymes involved in cleavages at paired basic residues in ACTH/endorphin cells of the anterior pituitary (which produces ACTH) and the corresponding cells of the intermediate pituitary (which cleaves ACTH to create aMSH and CLIP). Similarly, there are functionally distinct enzymes in enkephalin-producing cells and in dynorphin/neo-endorphin-producing cells (recall Fig. 1)42'4a. With only a couple of physiologically important exceptions (for example when insulin or parathyroid hormone secretion is suppressed), there is remarkably, little intraeellular degradation of peptides. Peptides are frequently stored for days after production in the same secretory granules in which the precursors were efficiently cleaved to the product peptides. Therefore it is reasonable to expect an enzyme, which is functioning in a test-tube like the biosynthetic cleavage enzyme in intact cells, to halt after the correct cleavages are completed. This criterion has not commonly been applied in evaluating the biological relevance of potential proteolytic processing enzyme preparations. Sometimes the order of cleavages within a precursor and the rates at which those cleavages occur can be exploited. For example, in the processing of proACTH/endorphin there is a rather rigid order of cleavages. First, /3-1ipotropin is cleaved from the precursor, and on a much slower time-scale /3-endorphin is liberated from /3-1ipotropin; seldom does the /3-endorphin get cleaved directly from the common precursor. By contrast, the cleavages of proinsulin to form insulin and C-peptide seem to occur at about the same time, with several biosynthetic intermediates present in comparable amounts 9.
TINS -June 1983
232 TABLE II. Selected amidated peptides and their precursor proteins, The amino acid sequences around known amidation sites are shown. The residue that will he amidated is tmdm'lined; adjacent Gly residues are circled; adjacent basic amino acids are doubly underlined. Hinge peptide (human and rat) and N-terminal glycopeptide (bovine or pig) are fragments of pro-ACTH/endorphin (see Fig. 1). References: hinge peptide4,',*°;melittinu; calcitonin2; vasopressin~; aMSH~; gastfin45;N.terminal glycopeptide
Peptide
Asn Glu
VIP Hinge peptide
-Pro-Arg-G..~..l~@
Gin
Melitlin
-Arg-Gln-_~ar ~31~ -OH
Pro
Calcitonin TRH Vasopressin LHRH Corticotropin-releasing factor Secretin c~MSH Gaswin-relensingpeptide PHI PYY Neuropcptide Y Pancreatic poly~ptide Gastrin
-'Gly-Ala-~ ~9-Lys-Lys-Arg-Asp-
Gly Ala Val Met lie Tyr
Phe
Precursor ? -L~s-Ar~-Ser-Tyr-
~
- Pro-Arg-Gly. ~,Lys-Arg-Ala-Met? ? -Lys-Pro-Val- ~9-Lys-Lys-A~.Arg. ? ? ? ?
N-terminal glycopeptide
There are clearly a large number of precursors to bioactive peptides, and there must also be at least several distinct proteolytic processing activities involved in the biosynthesis of the smaller peptides. The proteolytic enzymes seem to function in the endoplasmic reticulum and Golgi apparatus as well as in secretory granules. There are several distinct sequence spocificities exhibited by biosynthetic proteolytic enzymes;
help to solve some of these problems. There have been too many studies of proteolytic processing enzymes to provide a satisfactory overview of these data in this brief article; interested readers are referred to some recent reviewss,*s,3s,4~. Thel:e clearly remains a tremendous amount to be learned about biosynthetic proteolytic processing enzymes.
-Met-Asp-Lt~e- ~
-Arg-Arl~Ser-Al&
-Asp-Arg-Phe- ~
-Arg-Arg-Asn-Gly-
Both endopeptidases and exo~ptidases must he utilized; but there is not yet any clear idea of how many differentenzymes are involved. It is not known whether each peptide system has its own unique enzymes, or whether several generic enzymes accomplish most of the known cleavages. The ability to incorporate genes coding for these pro-rnolecdes into other cell types and follow their expression may
n Many bioactive poptides have carboxyl terminal o¢-amide residues (Table II). In general, presence of the a-amide is critical for biological activity: for example, the potency of corticotropin-releasing factor is diminished at least 1 000-fold when Ala replaces Ala-NI-k4. and deamidated analogs of gastrin and cholecystokinin are totally inactiveSt A variety of amino acids are amidated, but current data suggest that amino acids with charged side chains are amidated less frequently. In promelittin. the Gly following melittin is the carboxyl terminus of the molecule and is not followed by a pair of basic residues. In other cases where data are available, the precursor proteins to amidated peptides all contain the amino acid sequence -X-Gly-Basio.Basic- where X is the residue that becomes amidated in the mature peptide and the basic residues can be Lys or Arg. This presumed amidation plus proteolytic cleavage recognition site has been seen so universally that precursor proteins containing it (based on nucleotide analysis)
TABLE IlL Acetylation of aMSH and/3-endorphin Peptide
otMSH
Effect of a-N-acetylation on bioactivity 0
0
0
0
0
II
I[
II
H
t~
Nl-h-CH-C.Peptide ~ Cl-h-C-NH-CH-C-Peptide -* CI.-h-C-NH-CH-C-Peptide
I
t
I
CH2
CH2
CH2
OH
OH
O
Greatly increases skin-darkening potency
I
C=O
I
ACTH(I- 13)Nl-h
~-endorphin
a-N-acetyl- ACTH( 1-13)Nl-h
0
0
0
II
II
II
c¢-N,O-thacetyl-ACTH(l- 13)Nl-h
Nl-h.CH-C.-Peptide --~ Cl-h-C-NH-CH.C- Peptide
5 CI-G
© L
OH /3-endorphin(I-31)
CH,
© ~
Oil a- N-acetyl-~-endorphin( 1,3 i )
Greatly decreases opiate potency
233
T I N S - J u n e 1983
are often assumed to lead to a product peptide ending in -X-NFh (for example yl MSH, hinge peptide, calcitonin generelated peptide)2,4.t3. While such a sequence may be necessary for amidation to occur, it is not sufficient: the pro-ACTH/endorphin molecule contains the sequence - Val-Glys-Lys-Lys-Arg-Argwhich is converted into the VaI-NH2 of txMSH in intermediate pituitary cells but remains intact as the mid-region of ACTH(1-39) in anterior pituitary corticotropes. Tatemoto and MutP 9 noted that peptides with carboxyl terminal a-amides occur primarily among neural and hormonal peptides. Therefore they developed a chemical detection system for peptides containing carboxyl terminal a-amides. In this way they identified several previously unknown bioactive peptides. The same 27-residue secretin/glucagon/VIP/GIP-like peptide terminating in Ile-NH2 was found in extracts of pig intestine and brain. Two similar but distinct 36-residue pancreatic polypeptide-like peptides both terminating in Tyr-NH~ were found; one was localized. to brain and the other to intestine. Bradbury et al.5 recently succeeded in characterizing an amidating activity in extracts of pig pituitary secretory granules. This enzyme converts synthetic tripeptides ending in the sequence -X-GIy into the corresponding des-Gly peptide amide; X can be a neutral amino acid like Val, Phe or Gly, but not a charged amino acid like Asp or Lys. The nitrogen of the peptide amide comes from the a-amino group of Gly, and glyoxylate is released as a reaction product (Fig. 2). The substrate specificity of the pituitary enzyme suggests that the same enzyme could amidate aMSH, gastrin, cholecystokinin, oxytocin and vasopressin. The amidating ability of peptide-producing cells may show interesting regulatory properties: cultured dog pseudoislets continue to produce pro-pancreatic polypeptide and convert it into a pancreatic polypeptidesized molecule, but amidation no longer occurs after a period of time in cultureW"~7; similarly, although cultured rat intermediate pituitary cells produce aMSH-sized peptides, the peptides are no longer amidated and terminate with -Gly~4-OH.
Aeetylation Co-translational NH=-terminal acetylation of proteins is common, but its physiological role is not known. The posttranslational acetylation of histones on the e-Nl-h group of Lys has been extensively studied and may play a role in gene regulation. Among neural and hormonal peptides, post-translational Nl-h-terminal acetylation is an important modification for aMSH and
HOHHO
HO
I II I I II Peptide-C-C-N-C-C-OH I I R H residue to be
I II + Y
glycine
HO
I il
= Peptide-C-C-N=C-C-OH I R
+ YH2
unstable imine
amidated
HO
HO HO O0 I II I ,I li II Peptide-C-C-N=C-C-OH + H20------'~Peptide-C-C-NH2 + H-C-C-OH I I
I II
R
unstable imine
R
amidated peptide
glyoxylate
Fig. 2. Proposed mechanism for amidation. (Adapted from the reaction mechanism proposed by Bradbury et al. 5 The necessary oxidizing agent Y was not specified. )
/3-endorphin. In addition, about 5% of rat growth hormone=7 and 25% of Leuenkephalin in the neural lobe of the rat pituitary are NH2-terminally acetylated; interestingly, dynorphin, a 17-residue opioid peptide found in the hypothalamus and neural lobe of the pituitary, contains the Leu-enkephalin sequence at its Nl-hterminus but is not acetylated38. Given the dramatic effects of NHa-terminal acetylation on the bioactivity of aMSH, fl-endorphin and Leu-enkephalin, it will be of interest to look for this modification in other systems. Acetylation is one of the tissue-specific post-translational modifications involved in the processing of pro-ACTH/endorphin and occurs in the intermediate but not in the anterior pituitary (Table III). Acetylation of the NI-I~-terminus of ctMSH is required for potent melanocyte-stimulating activity while acetylation of/3-endorphin abolishes opiate activity; a single post-translational modification has evolved that activates one peptide (aMSH) while inactivating another (fl-endorphin). In the intermediate pituitary of several species, aMSH exists in three acetylation states: desacetyl-aMSH or ACTH(1-13) NI-h, aMSH or a-N-acetylACTH(1-13)NI-h, and a-N,O-diacetylACTH(1-13)NI-h7,15,sa. Pulse-chase labelling experiments have recently shown that the pathway for the acetylation of ACTHrelated molecules in the rat intermediate pituitary begins with desacetyl-aMSHsized molecules which are precursors to mono- and di-acetylated peptides15. There is some controversy regarding the acetylation of ACTH- and /3endorphin-related molecules in the brainx'2s.ss. Zakarian and Smyth*~ reported
finding primarily/3-endorphin(1-31) in the rat hypothalamus (the region containing fl-endorphin-producing nerve cell bodies) and acetylated forms in the hippocampus, colliculae and brain stem (primarily fiber terminal areas). Other studies have suggested the presence of only non-acetylated forms of fl-endorphin and aMSH in rat brain14. Non-specific acetylating activity has been found in homogenates of many tissues (for example pituitary, liver, lung, brain)3a.44; the enzyme activity responsible for the co-translational acetylation of proteins (probably associated with the rough endoplasmic reticulum of all tissues) is able to acetylate ACTH- and /3endorphin-related molecules in vitro. Biosynthetic labelling experiments coupled with subcellular fractionation of rat intermediate pituitary cells have shown that fl-endorphin(l-31 ), ACTH( 1-13)NI-h, and aMSH become NH~terminally acetylated when associated with secretory granules15. The secretory granules of the intermediate pituitary but not those of the anterior pituitary contain peptide acetyltransferase activity believed to be responsible for the acetylation of ACTH- and /3-endorphinrelated molecules in vivo. The secretorygranule-associated acetyltransferase from bovine intermediate pituitary acetylates ACTH(1-13)NI-h, aMSH and fl-endorphin(1-31). The acetylated forms of /3-endorphin inhibit the acetylation of both /3-endorphin and ACTH-related molecules and steady-state kinetic experiments have shown that this inhibition is competitivelY; this suggests that the same enzyme acetylutes both ACTH- and fl-endorphin-related peptides.
234
TINS - June 1983
TABLE IV. Sulfatedpeptides Phosphorylation The description of phosphorylated Peptide Sequencearoundsulfationsite Effect of sutfationon bioactivity ACTH and CLIP in rat pituitary marked the first time that naturally occurring small SOsH bioactive peptides were shown to be phost phorylateda.L In corticotropes of the rat Gastrin -Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Nl'h No effecton stimulationof gastric acid secretion anterior pituitary approximately half of the ACTH molecules are phosphorylated at SO,H SeP t , while in the rat intermediate pituitary Cholecystokinin . Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-Nl-h Essentialfor actionon gall bladder approximately two-thirds of the CLIP and pancreas molecules are phosphorylated. The degree SO3H of phosphorylation of ACTH and CLIP is 1 pyroGlu-Glrv Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-Nl-h species specific: mouse pituitary ACTH Caerulein and CLIP are phosphorylated 3-fold less S(hH I than the rat molecules; bovine pituitary Leu-enkephalin Tyr-Gly-Gly-Phe-Leu Inactivates optate bioactivity ACTH and CLIP are not phosphorylated; and about one-third of human ACTH is phosphorylateda, 12. Phosphorylation is a protein kinase- Sulfafian terminal N- acetylgalactosamine and mediated process which can be divided into Gastrin, cholecystokinin (CCK) and the N-acetylghtcosamine residues of the general mechanisms based on substrate amphibian peptid¢ ¢a~rdein represent a asparagine-linked oligosaceharide*'. This specificity. Cyclic nucleotide-dependent family of peptides derived from a common type of sulfation has been observed extenand calcium-dependent protein kinases ancestral peptide. The COOH-terminal sively in proteoglycan synthesiss° and phosphorylate serine residues located to the pentapcptid¢ of these hormones is identical recent evidence indicates that this mechanCOOH-terminal side of groupings of basic and this regionrepresents the active site of ism may be important for other pituitary amino acids 23, while the physiological both gastrin and CCK. In addition, the glycoproteins. Bovine thyrotropin and casein kinases phosphorylate Ser or Thr COOH-terminal region is also the site of human hiteinizing hormone also appear to residues where the sequence 1S sulfation for then~ peptides (Table IV). be sulfated like bovine luteiuizing hor-Ser(Thr)-X-Acidic-3t Phosphorytation of As reviewed by Rehfetd's, CCK is mone, while human chorionic gonadotrorat ACTH and CLIP occurs at the sequence synthesized by cells of the intestine and pin is not sulfated. Sulfation does not affect -Seraa-Ala3'-Glu3a-; in bovine ACTH, brain as part of a precursor protein consist- the ability of these hormones to bind to Glu 3a is replaced by Gin s3 and phosphoryla- ing of approximately 130 amino acids. Tim target tissue, however it is speculated that tion does not occur. This specificity thus precursor ,s successively cleaved to yield sulfated terminal sugars may function like resembles that of the physiological casein CCK-39, CL-'K-33, CCK-12, CCK,8 and negatively charged sialic acid residues to kinases. The rat pro-ACTH/endorphin CCK-4. All of the forms of CCK except enhance the half-lives of these hormones. molecule has potential phosphorylation CCK-4 arc sulfated and this modification is Rat pro-ACTH/endorphin may be sulfated sites in the Nl-h-terminal glycoprotein essential for stimulating gall.bL_a,~_~ empin a similar fashion *°. (Se# 8) and in o~MSH (Sera of ctMSH); tying and secretion of pancreatic enzymes. phosphorylatcd c~MSH has not been obGastrin is synthesized by cells of the A c k n o ~ n l s We thank out many colleaAgueswho provided served and the NH~.terminal glycoprotein stomach as part of a 105-amino-acid prepreimnts of their work and apologize for our inais phosphorylated to a much lesser extent cursor protein which is cleaved in a series of bility to cover all of the vast amount of relevant than ACTH or CLIP TM. Thus the sequence. steps to gastrin-34 and then to gastrin- 17. In material in this review. Research was supported Ser-X-Acidic- appears to be just part of the contrast to CCK, only approximately half by NIH grants AM-18929, AM-19859, signal for initiating phosphorylation. of the g a s ~ r e l a t e d forms are sulfated and NS-06648 and NS-06363, and a grant from the Studies of biosynthesis indicate that sulfation has no effect on the ability of McKnight Foundation, phosphorylation occurs shortly after syn- gastrin- 17 to stimulate gastric acid secrethesis of p r o - A C r H / ~ , l~-sama~y tion in mammals. It is not clear when during Reading list 1 Akil, H.. Shiomi.H.. Walker.J. M. and Watson. in the endoplasmic reticulum or Golgi at biosynthesis gastrin or its precursor is S. (1982)Adv. Biochem. PsychopharmacoL roughly the time that glycosylation of the sulfated. 33, 61-67 precursor is occurring. The two processes In addition to the gastrin-CCK related 2 Amara. S. G.. Jonas. V.. Rosenfeld. M. G., Ong, probably occur sequentially (glycosylation, peptides, Len-enkephalin with an OE. S. and Evans,R. M. (1982)Nature (LondonJ 298. 240-244 then phosphorylation) and neither seems to sulfated tymsyt residue has been isolated 3 Bennett, H. P. J., Browne, C. A. and Solomon,S. greatly affect the biological activity of from brain*'. Sulfated Lcu-enkephalin (1981) Proc. Nail Acad. Sci. USA 78. ACTH s, t,. represents 10-20% of the L e w e n k ~ 471"3-4717 Many other neural hormonal polypep- isolated from strintam and is biologically 4 Boileau, G,, Laraviere. N., HsL K.-L., Seidah. tides have potential sites for this type of inactive. At present it is not known wl,~hex N. G. and Chretien,M. (1982)B/ochem/stry21. phosphorylation. For example, numbering any of the other many enkephalin-related 5341-5346 with the first residue of the pro-molecule as peptides are sulfated. 5 Bradbury, A. F.. Finni¢, M. D. A. and Smyth, D. G. (1982)Nam.~ (London) 298,686-.688 No. 1, phosphorylation could occur at Very little is known about the sulfoffans6 Brown, M.. Rivier. J. and Vale. W. (1981) Ser ~ of pig pro-gastrin~; at Se# °+~t femse activities that must be involved in Endocrinology t08.2391-2393 and ThP"s of rat pro-calcitonin (assuming a linking sulfate to these specific tyrosyl 7 Browne,C. A., BenncR.H. P. J. and Solomon,S. 22-amino-acid signal peptide)'; at residues. (1982)Aria/. Biochem. 124,201-208 Sera''lS'''a and Thr s¢'*~s of bovine proBovine luteinizing hormone occurs in 8 CocbcR,A. D., Paterson.S. J.. MeKnight,A. T.. enkephalin in AS'; at S e t as'Ts't~'*6s'~tt of sulfated and non, sulfated forms, but in Magnan,J. and Kos~flitz, H. W. (1982)Nature pig pro-enkephalin B tt. (London) 299, 79-81 this case the sulfate is linked to the
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Richard E. Mains, Betty A. Eipper, Christopher C. Glembotski and Robert M. Dores were, at the time o f writing this review, at the Department of Physiology, C-240, University o f Colorado Health Sciences Center, 4200 East 9th Avenue, Denver, CO 80262, USA. Richard E. Mains and Betty A. Eipper are now at the Department o f Neuroscience, Johns Hopkins University School o f Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA. Christopher C. Glembotski is now at the Department o f Pharrnacology, University o f Pennsylvania School o f Medicine, 36th and Hamilton Walk, Philadelphia, PA 19104, USA. Robert M. Dores is now at the Mental Health Research Institute, University o f Michigan School of Medicine, Ann Arbor, M148109, USA.