sign in sign up
<<
Home , Carboxypeptidase B, Leucyl aminopeptidase, Prolyl aminopeptidase, Signal peptidase

CORE Metadata, citation and similar papers at core.ac.uk

Provided by Elsevier - Publisher Connector

Volume 234, number 2, 251-256 FEB 06071 July 1988

Review Letter

Proline residues in the maturation and degradation of hormones and neuropeptides

Rolf Mentlein

Anatomisches Institut der Christian-Albrechts Universitiit zu Kiel, OlshausenstraJe 40.2300 Kiel I, FRG

Received 4 May 1988; revised version received 23 May 1988

The involved in the maturation of regulatory like those of broader specificity normally fail to cleave peptide bonds linked to the cyclic . This generates several mature peptides with N-terminal X-Pro- sequences. However, in certain non-mammalian tissues repetitive pre-sequences of this type are removed by specialized dipeptidyl (amino)peptidases during maturation. In , proline-specific proteases are not involved in the biosyn- thesis of regulatory peptides, but due to their unique specificity they could play an important role in the degradation of them. Evidence exists that dipeptidyl (amino)peptidase IV at the cell surface of endothelial cells sequesters circulating peptide hormones which arc then susceptible to broader attack. The cleavage of several neuropeptides by prolyl has been demonstrated in vitro, but its role in the brain is questionable since the precise localiza- tion of the is not clarified.

Neuropeptide; Peptide hormone; Proteolytic processing; Degradation; Dipeptidyl (amino)peptidase;

1. INTRODUCTION 2. DIRECTION OF THE PROTEOLYTIC MATURATION OF REGULATORY The unique cyclic and imino structure of the PEPTIDES BY PROLINE RESIDUES amino acid proline influences not only the confor- mation of peptide chains, but also restricts the at- Regulatory peptides are synthesized as part of tack of proteases. Peptide bonds involving proline larger precursors, namely the prepro-peptides. The residues are often resistant to the action of endo- N-terminal pre-sequence is cleaved by a signal pep- and [l-4], even those with broad tidase in the rough [5-81. specificity (table 1). On the other hand, several Further proteolytic maturation occurs in the Golgi proteases are known which specifically or selective- apparatus and in the secretory vesicles. The key ly attack proline bonds (table 2). This report sum- proteolytic for the processing of pro- marizes some recent findings about the influence peptides include [7,8] an endopeptidase cleaving at of proline residues (i) on the proteolytic processing paired basic residues, further a of neuropeptides and peptide hormones from their B-like and/or an -like precursors, and (ii) on the final degradation of enzyme for the removal of C- and N-terminal ex- these regulatory peptides. tended basic residues from the fragments. Other proteases [7-91 as well as further nonproteolytic modifications like glutaminyl cyclisation [lo], Correspondence address: R. Mentlein, Anatomisches Institut der Christian-Albrechts Universitat zu Kiel, Olshausenstr. 40, acetylation, amidation, glycosylation [6-81 may 2300 Kiel 1, FRG also be necessary, depending on the complexity of

Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/88/$3.50 0 1988 Federation of European Biochemical Societies 251 Volume 234, number 2 FEBS LETTERS July 1988

Table 1

Peptide bonds stabilized against proteolytic attack by some common proteases by involvement of proline residues

Protease Peptide bonds ( -), normally cleaved if Pro is replaced by other amino acids

Trypsin - (Arg, Lys) - Pro - - (Trp, Tyr, Phe, Leu, others) - Pro - - (many) - (hydrophobic) - Pro - Staphylococcus aweus protease - (Glu, Asp, Gin, Asn) - Pro - Aminopeptidase M” (= microsomal alanyl (neutral) - Pro - aminopeptidase) Pro - (many) - (hydrophobic) - Pro - A and B - (many) - Pro B2 (= lysosomal ) - Pro - many Angiotensin converting enzyme (= peptidyl A) - (many) - Pro - (many)

Data compiled from [l-3]. Reports about a different specificity of aminopeptidase M seem to be caused by contaminations with other exopeptidases, e.g. IV [4] the maturation. Fig. 1 illustrates the stability of X- The substance P precursor is not cleaved behind Pro- bonds in the precursors of some regulatory the dibasic sequence -Arg-Arg-Pro- because a pro- peptides to the processing proteases (further ex- line residue follows. Instead, the cleavage occurs amples [9]). between the two basic residues Arg-57 and Arg-58

Table 2

Proline specific mammalian endo- and exopeptidases

Enzyme Requirements of the cleaved peptides M, (x 10-3) Inhibitors PH Locali- optimum zation Cleavage Other Residues Chain length Native Subunit site residues accepted (no. of . = Pro accepted at 0 residues) at 0

Prolyl endopeptidase & _ #Pro 4-17 70” 74a Dip-F 7.5 S(M) Prolyl amino- peptidase 0-k _ hydrophobic 2-4 300” W EDTA 8.1 S Aminopeptidase P CA- HYP f Pro 3-9 1, IO-phen- anthroline 7.8 M Dipeptidyl pepti- & dase II Ala any 3-11 lloa 53” Dip-F, cations 5.5 S(M) Dipeptidyl peptidase IV ++l__ Ala, Hyp #Pro#Hyp 3-200 220b 120b Dip-F, bacitracin 8.0 Lysosomal prolyl carboxypeptidase 40 _ #Pro, hydrophobic 23 210c 2sc Dip-F 5.2 Carboxypeptidase P --dc Ala, Gly #Pro 2-7 240’ 7.8 Prolinase dc HYP any 2 300’ EDTA,Cd’+- activated 8.7 Prolidase CA HYP #Pro 2 108’ 54’ EDTA, mer- curibenzoate 7.5

The data are compiled from [2] and from work in own laboratory; for action on regulatory peptides see text. S, soluble; M, membrane- bound; molecular data for the rat brain enzymesa, rat kidney enzymesb and kidney enzymes’; @, any unprotected amino acid; Dip-F, diisopropyl fluorophosphate; all peptidases sensitive to chelating agents and carboxypeptidase P are activated by Mn2+

252 Volume 234, number 2 FEBS LETTERS July 1988

PROCESSED SEQUENCE MATURE PEPTIDE (Ref.1

. . . Ala - Arg ____-l Arg - Pro - Lys - Pro... Substance P [ 11 1

. LYS - Lys _____+ Q-IA,, - Pro - Val .,. CLIP (121

. . . Arg - LYS _____+ a- Arg - Pro - Tyr .._ h a-Relaxin (13)

4 . Ah - Gin - Gly -____ Ala - Pro - Leu . . h Pancreatic Polypeptide (1.4)

4 . . . Asn- Ser --___ Ala - Pro - Thr . . h lnterleukln II II5 1

4 . . . His - Ser _____ Ala - Pro - Asp . h a-Chorionicgonodotropin 15)

Fig. 1. Influence of proline residues on the cleavage of precursors of mgulatory peptides by mammalian processinglproteases (see text). Observed cleavage by basic residue-specific P (o = Arg, Lys), signal peptidase w (o = Ala and others), and aminopeptidasess . The N-terminal sequences of the mature peptides are boxed.

of the precursor yielding a N-terminal Arg-Pro-. . . been found to be either Ala, Ser, Gly, Cys, Thr or sequence in the mature bioactive peptide. This pro- Gln [5,6]. From the examples in fig.1 it appears teolytic conversion has earlier been classified as that the removal of the pre-sequences may also be ‘proline-directed (monobasic) arginyl cleavage’ influenced by proline residues. In these examples [9]. A more complex example is the conversion of all Ala-Pro- sequences are resistant to the signal ACTH to wMSH (= acetylated and amidated peptidase, and instead, cleavage occurs after Ser or ACTH l- 13, melanocyte-stimulating hormone) Gly which, of course, are also possible cleaving and CLIP (= ACTH 18-39, corticotropin-like in- sites. Nevertheless, a list of 90 cleavage sites for termediate lobe peptide) in the pituitary in- signal peptides gives no example of X-Pro termediate lobe. These bioactive fragments are [5]. linked by four basic amino acid residues. As con- In conclusion, the proteases involved in the in- cluded from the occurrence of intermediates [12], tracellular precursor processing of bioactive pep- the endopeptidase initially cleaves ACTH( l-39) at tides normally are unable to cleave X-Pro- bonds. the Lys-16-Arg-17 bond. By removal of one However, especially in invertebrates and lower residue, ACTH(17-39) is then processed vertebrates processing enzymes with substrate to CLIP by the action of an aminopeptidase. specificities to overcome this restriction must exist. However, the Arg-18 residue is stable to further The pro-sequence of honey bee promellitin consists aminopeptidase sequestration, because a proline of six X-Pro and five X-Ala repetitive dipeptidyl residue follows. The same mechanism should oc- residues [16] which should be removed by the se- cur in the processing of the human relaxin a-chain quential action of a dipeptidyl peptidase of this precursor. As concluded from these data, basic specificity to yield the mature mellitin. The pro- residue-specific endopeptidases and aminopep- sequence of the antifreeze from the winter tidases fail to cleave -Arg-Pro- sequences in the flounder contains four X-Pro and seven X-Ala maturation of peptide precursors. This may ex- repetitive sequences [17], but in this case the pro- plain the generation of the non-processed sequence cessing to the mature peptide occurs in serum [ 181. -Lys-Pro-Arg-Arg-Pro- in mature neurotensin. Further investigations are required to clarify Also the carboxypeptidase B-like enzyme cannot whether the stepwise cleavage of dipeptides in- readily remove Arg from -Pro-Arg peptide cluding X-Pro- sequences [19] is a particular fragments [9]. mechanism for the intracellular maturation of pro- The last residue of the signal (pre-) sequence has peptides also in mammals.

253 Volume 234, number 2 FEBS LETTERS July 1988

3. PROLINE-SELECTIVE PROTEASES IN of the N-terminus with and without the X-Pro- se- THE DEGRADATION OF MATURE quence. Since this heterogeneity does not result PEPTIDES from intracellular processing [28], the action of DPP IV on the circulating hormones may be The occurrence of proline-selective proteases, responsible for it. However, with the exception of which cleave peptide bonds resistant to most other substance P [29] and possibly His-Pro-NH2 [22] proteases, suggests that these specialized enzymes the loss of the terminal dipeptidyl sequences does could play a key role in the final degradation of not decisively inactivate these peptides. Rather, the mature peptide hormones or neuropeptides. Beside DPP IV action might facilitate the further prolyl endopeptidase, the proline-selective dipep- degradation, since the sequestered residue is then tidy1 peptidases are the most attractive candidates susceptible to subsequent aminopeptidase attack for such a function since several bioactive peptides [4]. As for other neuropeptide degrading pro- start with N-terminal X-Pro- sequences as dis- teases, the biological function of DPP IV in brain cussed above. Like N-terminal cyclisation to depends on its precise localization. DPP IV is pre- pyroglutamic acid, acetylation or C-terminal sent at the surface of brain capillaries and lep- amidation, these sequences with a penultimate pro- tomeninges [30,31], and has also been detected in line could be regarded as being protected against synaptosomal fractions [22,32]. In the developing the degradation of bioactive peptides by non- brain DPP IV is abundant in neuroblasts [30]. The specialized exopeptidases. sequestration of the N-terminal tetrapeptide Arg- Indeed, human dipeptidyl peptidase IV (DPP’ Pro-Lys-Pro of substance P in neuronal cell IV, EC 3.4.14.5) liberates X-Pro-dipeptides with cultures [33], which stimulates neurite outgrowth high activity from several regulatory peptides of [34], shows a possible importance of DPP IV in various chain lengths [20,21]: e.g. from His-Pro- neuronal proliferation. NH2 (thyroliberin-fragment generated by pyroglu- A further proline-selective dipeptidyl peptidase, taminase, of which the metabolite His-Pro 1221 or DPP II (EC 3.4.14.2), is a lysosomal enzyme oc- the non-enzymatic formed cyclo(His-Pro) may be curring in endocrine glands, reproductive organs bioactive), &casomorphin (opioid peptide from and neurons of the brain [2]. It liberates X-Pro- as milk, Tyr-Pro-Phe-Pro-Gly-Pro-Be), substance P well as X-Ala-dipeptides only from short peptides (Arg-Pro-Lys-Pro-Gln.. . , Mr 1348), pancreatic up to 11 residues long [35], even if followed by a polypeptide (Ala-Pro-Leu.. . , M, 4240), prolactin further proline. Because of this localization and (Thr-Pro-Val.. . , A4, 24000) or human chorionic specificity it is suggested that DPP II accomplishes gonadotropin (a-chain Ala-Pro-Asp.. . , M, the final degradation of fragments from interna- 48000). Only peptides with proline in the third lized or over produced regulatory peptides. Se- position, e.g. bradykinin (Arg-Pro-Pro.. .), or quences involving proline bonds are stable to most glycosylation here, e.g. interleukin II (Ala-Pro- lysosomal proteases. The dipeptides liberated by Thr(CH0). . .) or ai-microglobulin (Gly-Pro-Val- the action of DPP II (in contrast to tri- and Pro-Thr-(CHO). . .) are protected against DPP IV oligopeptides) readily cross the lysosomal mem- attack [21]. Since DPP IV is found in high concen- brane, and then are metabolized by the ubiquitous trations at the vascular surface of the capillary en- cytosolic prolidase (table 2). N-terminal X-Pro- se- dothelium [23] and the kidney brush border [2], quences may also be overcome by the sequential circulating peptide hormones of such structure action of two specialized , namely should be rapidly sequestered by this enzyme when aminopeptidase P and (or entering the blood stream and/or during renal other aminopeptidases of broader specificity). passage. Though no direct experiments, e.g. by use Aminopeptidase P (= microsomal proline amino- of specific DPP IV inhibitors, have yet been under- peptidase, EC 3.4.11.9) is a membrane-bound en- taken, some findings are consistent with this zyme with widespread tissue distribution [2,36]. In hypothesis. Sequence data for the a-subunit of the lung it initiates the degradation of circulating chorionic gonadotropin [24], follicle-stimulating bradykinin [37]. This vasoactive peptide is com- hormone 1251, luteinizing hormone [26] or thyroid- pletely inactivated during a single passage through stimulating hormone [27] exhibit a heterogeneity the rat lung. After liberationof the N-terminal Arg

254 Volume 234, number 2 FEBS LETTERS July 1988 by aminopeptidase P, the resulting octapeptide is precisely, additional data about its tissue localiza- easily sequestered by combined action of DPP IV tion and specificity are needed. and aminopeptidase M [37]. Prolyl aminopep- The proline specific , namely pro- tidase (EC 3.4.11.5) has been detected as a lidase ( = proline dipeptidase, EC 3.4.13.9) cleav- melanostatin (Pro-Leu-Gly-NHz) degrading en- ing X-Pro and prolinase (= prolyl dipeptidase, EC zyme [1,2] in the kidney, It has not been in- 3.4.13.8) cleaving Pro-X dipeptides, are cytosolic vestigated whether this enzyme attacks strictly this enzymes of widespread tissue distribution [ 1,2]. substrate or shows broader aminopeptidase prop- Both are strict dipeptidases, and exhibit a complete erties. (prolidase) or high (prolinase) specificity for pro- Prolyl endopeptidase (= post-proline cleaving line or hydroxyproline. Pro-X dipeptides may also enzyme, post-proline endopeptidase, TRH- be hydrolysed by dipeptidases of broader specifici- deamidase, brain-kinase B, endooligopeptidase B, ty [l], or by prolyl aminopeptidase [2]. Due to EC 3.4.21.26) specifically cleaves the -Pro-X- their localization, both dipeptidases are considered bond (but not -Pro-Pro-) in several neuropeptides: to metabolize proline-containing peptide frag- oxytocin, vasopressin, gonadoliberin, cu-MSH, ments produced by lysosomal or other intracellular substance P, neurotensin, thyroliberin, bradykinin proteases. Inherited prolidase deficiency results in and angiotensin II [38]. High activities of prolyl a massive urinary excretion of X-Pro dipeptides, endopeptidase are found in the brain. It seems to especially Gly-Pro, and mainly affects collagen be mainly a soluble, probably cytosolic protein, with clinical signs of lathyrism [2]. and therefore its role in the initial degradation of This reflects the important role that proline- secreted neuropeptides is questionable. The specific proteases also have in the degradation of specific function(s) of this enzyme may be clarified structural , especially collagen and elastin. by the use of the selective inhibitors, Z-Pro- But this is another topic. prolinal [39] or Z-Gly-Pro-diazomethylketone [40]. However, a prolyl endopeptidase cleaving the Pro( IO)-Tyr( 11) bond of neurotensin has recently 4. PERSPECTIVES been purified from rat brain synaptic membranes [41]. Further studies of the precise cellular and Due to their unique specificity proline-specific subcellular localization in brain are required to proteases have been investigated by several en- evaluate the role of prolyl endopeptidases in zymologists. However, the functions of these en- neuropeptide metabolism or other neuronal zymes are still far from being resolved. Depending functions. on their localization, namely extracellular (at cell Several mammalian carboxypeptidases are surface) or intracellular (at several sites), proline restricted in their action by the presence of proline selective proteases may play a role in several pro- in the C-terminal ultimate and/or penultimate tein degradative systems. To clarify their role in position (table 1). Proline-specific carboxypep- the metabolism of regulatory peptides, future tidases are the lysosomal prolyl carboxypeptidase studies should be directed to examine their precise (= angiotensinase C, EC 3.4.16.2) and the localization, especially in the brain, and to develop microsomal prolyl carboxypeptidase ( = carboxy- further specific inhibitors which could be applied peptidase P, EC 3.4.17) which both liberate the to cell cultures or in vivo. terminal hydrophobic amino acids after the penul- timate proline. The former enzyme has been con- Acknowledgements: I thank the Deutsche Forschungsge- sidered to be important for regulating angiotensin meinschaft (grant Me 758/2-l) for support, and Eberhard levels in kidney [42]; however, its distribution in Heymann and Gernot Struckhoff for stimulating discussions. diverse cells such as macrophages, endothelial cells and fibroblasts suggests a broader role in in- tracellular protein degradation [2]. The kidney REFERENCES microsomal prolyl carboxypeptidase may extend the high proteolytic activity of the renal brush [l] Walter, R., Simmons, W.H. and Yoshimoto, T. (1980) border membrane [43]. To define its role more Mol. Cell. Biochem. 30, 111-127.

255 Volume 234, number 2 FEBS LETTERS July 1988

[2] McDonald, J.K. and Barett, A.J. (1986) Mammalian 1231 Palmieri, F.E. and Ward, P.E. (1983) Biochim. Biophys. Proteases: A Glossary and Bibliography, ~01.2. Acta 755, 522-525. Exopeptidases, Academic Press, London. [24] Bellisario, R., Carlsen, R.B. and Bahl, O.P. (1973) J. [3] Croft, L.R. (1980) Handbook of Protein Sequence Biol. Chem. 248, 6796-6809. Analysis, 2nd edn, p.10, Wiley, Chichester. [25] Rathman, P. and Saxena, B.B. (1975) J. Biol. Chem. 250, [4] Heymann, E. and Mentlein, R. (1986) J. Dairy Res. 53, 6135-6146. 229-236. [26] Liu, W.-K., Nahm, H.S., Sweeny, C.M., Lamkin, W.N., [5] Von Heijne, G. (1983) Eur. J. Biochem. 133, 17-21. Baker, H.N. and Ward, D.N. (1972) J. Biol. Chem. 247, [6] Richter, D., Ivell, R., Schmale, H., Nahke, P. and 4351-4364. Krisch, B. (1985) in: Neurobiochemistry - 36. [27] Liao, T.H. and Pierce, J.G. (1971) J. Biol. Chem. 246, Colloquium Mosbach 1985, pp.33-42, Springer, Berlin. 850-865. [7] Gainer, H., Russel, J.T. and Loh, Y.P. (1985) [28] Birken, S., Fetherston, J., Desmond, J., Canfield, R. and Neuroendocrinology 40, 171-184. Boime, J. (1978) Biochem. Biophys. Res. Commun. 85, [8] Loh, Y.P. and Parish, D.C. (1987) in: Neuropeptides and 1247-1253. Their Peptidases (Turner, A.J. ed.) pp.65-84, VCH [29] Blumberg, S. and Teichberg, V.I. (1982) in: Regulatory Verlagsgesellschaft, Weinheim. Peptides: From Molecular Biology to Function (Costa, E. [9] Schwartz, T.W. (1986) FEBS Lett. 200, l-10. and Trabucchi, M. eds) pp.445-452, Raven, New York. [lo] Fischer, W.H. and Spiess, J. (1987) Proc. Natl. Acad. Sci. [30] Bernstein, H.-G., Schon, E., Ansorge, S., Rose, I. and USA 84, 3628-3632. Dorn, E. (1987) Int. J. Dev. Neuroscience 5, 237-242. [ll] Nawa, H., Hirose, T., Takashima, H., Inayama, S. and [31] Mitro, A. and Lojda, Z. (1988) Histochemistry 88, Nakanishi, S. (1983) Nature 306, 32-36. 645-646. [12] Fenger, M. (1986) Biochem. J. 235, 715-722. [32] O’Connor, B. and O’Cuinn, G. (1986) Eur. J. Biochem. [13] Hudson, P., Haley, J., John, M., Cronk, M., Crawford, 154, 329-335. R., Haralambidis, J., Tregear, G., Shine, J. and Niall, H. [33] Horsthemke, B., Schulz, M. and Bauer, K. (1984) Bio- (1983) Nature 301, 628-631. them. Biophys. Res. Commun. 125, 728-733. 1141 Takeuchi, T. and Yamada, T. (1985) Proc. Natl. Acad. 1341 Narumi, S. and Maki, Y. (1978) J. Neurochem. 30, Sci. USA 82, 1536-1539. 1321-1326. [15] Taniguchi, T., Matsui, H., Fujita, T., Takaoka, C., [35] Mentlein, R. and Struckhoff, G. (1988) Eur. J. Biochem., Kashima, N., Yoshimoto, R. and Hamuro, J. (1983) in press. Nature 302, 305-309. [36] Holtzman, E.J., Pillay, G., Rosenthal, T. and Yaron, A. [16] Lane, C.D., Champion, J., Haiml, L. and Kreil, G. (1987) Anal. Biochem. 162, 476-484. (1981) Eur. J. Biochem. 113, 273-281. [37] Orowski, A.T., Susz, J.P. and Simmons, W.H. (1987) [17] Davies, P.L., Roach, A.H. and Hew, C.-L. (1982) Proc. Mol. Cell. Biochem. 75, 123-132. Nat]. Acad. Sci. USA 79, 335-339. [38] Wilk, S. (1983) Life Sci. 33, 2149-2157. [18] Hew, C.L., Wang, N.C., Yan, S., Cai, H., Sclater, A. [39] Chu, T.G. and Orlowski, M. (1984) 23, and Fletcher, G.L. (1986) Eur. J. Biochem. 160,267-272. 3598-3603. [19] Mollay, C., Vilas, U., Hutticher, A. and Kreil, G. (1986) [40] Knitsatschek, H. and Bauer, K. (1986) Biochem. Biophys. Eur. J. Biochem. 160, 31-35. Res. Commun. 134, 888-894. [20] Piischel, G., Mentlein, R. and Heymann, E. (1982) Eur. [41] Checler, F., Vincent, J.-P. and Kitabgi, P. (1986) J. Biol. J. Biochem. 126, 359-365. Chem. 261, 11274-11281. [21] Nausch, I. (1987) Thesis, Frankfurt. [42] Oyada, C.E., Marinkovic, D.V., Hammon, K.J., [22] Coggins, P. J., McDermott, J.R., Snell, C.R. and Gibson, Steward, T.A. and Erdos, E. (1978) J. Biol. Chem. 253, A.M. (1987) Neuropeptides 10, 147-156. 5927-593 1. [43] Kenny, J. (1986) Trends Biochem. Sci. 11, 40-42.

256