Post-secretory processing of regulatory peptides: The pancreatic polypeptide family as a model example

Biochimie (1994) 76, 283-287

© Soci6t6 fran~aise de biochimie et biologic mol6culaire / Elsevier, Paris

283

Post-secretory processing of regulatory peptides: The pancreatic polypeptide family as a model example MS Medeiros, AJ Turner* Department of Biochemisoy and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

(Received 18 February 1994; accepted 31 May 1994)

Summary - - Post-secretory metabolism is an important event in the overall homeostasis of regulatory peptides and the enzymes involved in these processes may be suitable targets for pharmacological intervention. Some examples are reviewed here. Peptide YY and neuropeptide Y, both members of the pancreatic polypeptide family, can be processed by dipeptidyl peptidase IV to their (3-36) fragments by removal of the N-terminal Tyr-Pro dipeptide, which generates a metabolite of different receptor selectivity. Aminopeptidase P and endopeptidase-24,l I also metabolize these peptides and the relative levels of these three cell-surface enzymes may regulate their interconversionbetween receptor-selective forms and inactive metabolites. peptide YY / neuropeptide Y / pancreatic polypeptide / metallopeptidases / phosphoramidon / dipeptidyi peptidase IV

Introduction The major site of processing of peptide hormone precursors is in the secretory granule where prohormone convertases cleave specifically at mono- and di-basic sites within the precursor [1]. Additional processing through~ for example, C-terminal amidation and carboxypeptidase action produces the final, secreted form of the peptide. In some cases, however, the released peptide may need to be further processed to generate full biological activity, either at the cell surface or in plasma. A classical example is provided by the reninangiotensin cascade in which angiotensinogen is first converted by circulating renin to angiotensin I and then further processed by angiotensin converting enzyme, located on the surface of lung endothelial cells,: to the vasoactive peptide, angiotensin II [2]. There are also examples where post-secretory processing can convert a peptide hormone to a molecule of differing physiological activity, for example in the production of eosinophil chemotactic peptide from calcitonin gene-related peptide [3] or 'mini-glucagon' (glucagon [19-29]) from glucagon itself [4]. It is rapidly becoming clear that the post-secretory metabolism of peptides is an important event in the overall homeostasis of peptides [5] and the novel enzymes involved in these processes may be suitable targets for

*Correspondence and reprints

pharmacological intervention. The present article will address one example of such secondary processing, namely the processing of members of the pancreatic polypeptide family, especially peptide YY (PYY) and neuropeptide Y (NPY), which can be converted, through the action of cell-surface enzymes, to peptides of different receptor selectivity. NPY and PYY are 36 amino acid regulatory peptides which show high structural homology to each other and are also related to pancreatic polypeptide [6]. NPY is widely distributed in the nervous system and displays a large range of functions such as stimulation of appetite, anxiolysis/sedation, memory processing and modulation of pituitary hormone release. It is also known for its peripheral vasoconsmctor action. PYY is localized in endocrine cells of the gastrointestinal mucosa and has a variety of actions in the digestive system, all of an inhibitory nature [7]. Three distinct receptor sub-types have been described for NPY and PYY: Y1 receptors require the full length 36 amino acid peptide for their binding whereas Y2 receptors are also able to bind to C-terminal truncated forms. In particular, the (3-36) fragment of these peptides is found in the circulation and is highly selective for the Y2 receptor [8, 9]. The Y3 receptor shows high affinity NPY sites but does not bind PYY. Here we have compared the processing and metabolism of NPY and PYY by brush border membrane preparations and by some purified cell-surface peptidases.

284 ( des T, rr I -NPY

NPY

[ d n T y r -Pro] - NPY

I

°"

T

Tyr -Pro

A,~ Tyr

i i

I

TIME ( mini

"l

~

[

l

TIME ( mini

TIME I rain I

Fig 1. HPLC analysis of the metabolism of NPY by NEP, AP-P and DPP-IV. Human NPY (50 pM) was incubated with purified peptidases for 2 h at 37°C. Products were resolved by HPLC as described in Materials and methods. Metabolism is shown by: a, NEP (100 ng protein); b, AP-P (2 lag protein); and e, DPP-IV (500 ng protein).

APP-P

DPP-IV

$

$

H-Tyr-Pro-Ile-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-AsnArg-Tyr-Tyr-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-TyrNH2 Fig 2. Cleavage sites identified in peptide YY from the actions of aminopeptidase P (AP-P), dipeptidyl peptidase-IV (DPP-IV) and endopeptidase-24.11 (NEP).

M a t e r i a l s a n d methods Human P i'Y and NPY were obtained from Bachem UK Ltd, Essex, UKo Phosphoramidon was obtained from Peninsula Laboratories Europe Ltd, St Helens, UK. Other reagents were from Sigma, Poole, Dorset, UK. Endopeptidase-24.11 (neutral endopeptidase; NEP, EC 3.4.24.11), dipeptidyl peptidase IV (DPP-IV; EC 3.4.14.5) and aminopeptidase P (AP-P; EC 3.4. I ! .7) were purified to homogeneity from pig kidney cortex. Each protein exhibited a single polypeptide band on SDS-polyacrylamide gel electrophoresis. The purification of NEP was achieved by immunoaffinity chromatography [10] employing the monoclonal antibodies GK~tA9 respectively. Human brush border membranes were prepared by (he method of Booth and Kenny [ 1 ! ] and Kessler et al [ 12]. Human PYY or NPY (50 laM) were incubated individually with purified peptidases (100 ng-2 pg protein) or with human renal intestinal (jejunal) microvilla~, membranes (5 lag protein) in the presence or absence of specific peptidase inhibitors in

0.1 M Tris-HC! buffer (pH 7.4), in a total volume of 100 pl at 37°C for 2 h. The products of peptide metabolism were analysed by reverse-phase HPLC, usi,g a pBondapak Cig column. The fractionation used a linear gradient of acetonitrile from 4.05-45% in 0.08% H3PO4 (pH 2.5), followed by 5 min isocratic elution at final conditions. The products were monitored at 214 nm. Protein concentrations were determined by the bicinchoninic acid (BCA) method [13], using bovine serum albumin as standard. Results

Metabolism of PYY and NPY by purified peptidases Both P Y Y and N P Y were susceptible to hydrolysis by the three purified m e m b r a n e peptidases, NEP, D P P - I V

285 and AP-P. Figure 1 illustrates the HPLC profiles for hydrolysis of NPY by these enzymes. Only a single cleavage site was detected for both AP-P and DPP-IV consistent with their known specificity, producing respectively NPY-(2-36) and NPY-(3-36). Similar products were detected from PYY (data not shown). Incubation of either PYY or NPY with NEP resulted in several cleavage sites and, in the case of PYY, the primary site of cleavage was identified as the Asn 29Leu30 bond, an inactivating cleavage. The Km value for NEP with PYY as substrate was determined as 29 laM. The sites of hydrolysis of PYY by the three enymes are summarized in figure 2.

Metabolism of NPY and PYY by brush border membranes When incubated with a preparation of human renal microvillar membranes (5 lag, 2 h), NPY and PYY were substantially degraded and the NEP inhibitor phosphoramidon (10 laM) inhibited total metabolism by approx 77% in the case of PYY (fig 3a, b) and by 57% in the case of NPY. The production of the N-terminal fragments, Tyr and Tyr-Pro, was detected from both peptides. Their formation was not affected by phosphoramidon. The production of Tyr-Pro was inhibited completely by the DPP-IV inhibitor di-isopropylfluorophosphate (Dip-F). The release of the N-terminal tyrosine is presumably due to the action of AP-P but there is no specific inhibitor of this enzyme available. When the metabolism of PYY by human jejunal brush border membranes was compared, phosphoramidon had a much lesser effect (fig 3) indicating that enzymes additional to NEP also contribute to PYY metabolism in this preparation. Tyr-Pro was again identified as a product and its formation was completely suppressed by Dip-E

Discussion Since PYY(3-36) is a highly selective Y2 receptor agonist present in the circulation [8, 9], this implies specific post-secretory processing of this peptide and a similar phenomenon appears to exist with NPY. The presence of a prolyl residue at position 2 in ".he sequence of both PYY and NPY suggests the likelihood of metabolism by two proline-specific cell-surface peptidases: DPP-IV and AP-E This has been confirmed with the purified peptidases and the N-terminal products, Tyr and Tyr-Pro, are also produced when the intact peptides are incubated with renal brush border membranes. The production of Tyr-Pro is completely inhibited by Dip-F which is consistent

with the action of DPP-IV since this is the only serine proteinase in the renal microvillar membrane. In labelling experiments with [32p]_ or [3H]-Dip-F on pig and human kidney membranes only one radioactive polypeptide of 130 kDa in each case was seen after SDS-PAGE [14, 15]. No selective inhibitor of AP-P is available to confirm its participation but only a single proline aminopeptidase has been identified in brush border membranes. NEP also efficiently processes PYY and NPY but the primary site of metabolism represents an inactivating cleavage. More than 85% of the metabolism of PYY by renal brush border membranes appears to be accounted for by the three enzymes NEP, AP-P and DPP-IV. In renal brush border membranes, NEP is the predominant activity metabolizing PYY and NPY but, at other cell-surfaces, eg on endothelial cells where NEP levels are extremely low [ 16], DPP-IV may play a much greater role. A recent report examining the metabolism of NPY and PYY by endothelial cells in culture would support this interpretation [17]. Exam~.a.tion of the possible co-localization of these membrane peptidases with the various classes of PYYNPY receptors in the gastro-intestinal tract and elsewhere, would be worthwhile exploring. In conclusion, post-secretory processing is emerging as an important aspect of the biosynthesis and further metabolism of peptide hormones and neuropeptides. In some cases, the processing results in the conversion of an inactive precursor to an active hormone, as is seen with the angiotensin system and with the conversion of 'big endothelin' to 'endothelin' [18], a process which is catalysed by a phosphoramidonsensitive activity resembling NEP [ 19]. The pancreatic polypeptide family represent a subtle example of such a mechanism in which N-terminal processing can produce ligands of differing receptor selectivity. Thus, the relative levels of NEE DPP-IV and AP-P at different cell surfaces may play a pivotal role in post-secretory processing of PYY and NPY to receptor-selective agonists or inactive metabolites. The development of selective and potent DPP-IV inhibitors may provide a route to modulate the relative levels of PYY and NPY and their truncated (3-36) fragments. Metalk~pe.ptidases are of considerable importance in the processmg and metabolism of regulatory peptides with NEP in particular inactivating a wide variety of such molecules. Candidate substrates fox"both AP-P and DPP-IV are much more limited and the pancreatic polypeptide family represents a new and important addition to this group.

Acknowledgment We should like to thank the Medical Research Council (UK) for their generous support of this work.

286

PYY O.

PYY(I-29)

C.

PYY

A21t

"JIUUI

'"

I I I ry,-Pro

l/ • I

:;; LL

I

LLJ 2o

TIME ( rain )

PYY

.

,.

b.

%1

T

A~la PR

t.

?

i3 '.°'o LL O,

5,

iO, . . . . . .15 .

2~) O TIME ( rain I

-v S

IO '

15 ,

20,

Fig 3. HPLC analysis of PYY metabolism by kidney or intestinal brush border membranes. PYY (50 pM) was incubated with kidney brush border membranes for 2 h at 37°C in the presence or absence of phosphoramidon (10 laM). Products were resolved by HPLC as described in Materials and methods. The metabolism of PYY is shown by" a, kidney brush border membranes (5 lag protein); b, kidney brush border membranes plus 10 laM phosphoramidon; c, jejunal brush border membranes (5 lag protein); and d, jejunal brush border membranes plus 10 pM phosphoramidon.

287

References 1 Seidah NG, Day R, Chr~tien M (1993) The family of pro-hormone and proprotein convertases. Biochem Sac Trans 21,685-691 2 ErdOs EG (1990) Angiotensin 1 converting enzyme and the changes in our concepts through the years. Hypertension 16, 363-370 3 Davies D. Medeiros MS, Keen JN, Turner AJ, Haynes LW (1992) Endopeptidase-24.11 cleaves a chemotactic factor from o~-calcitonin gene-related peptide. Biochem Pharmaco143, ! 753-1756 4 Pavoine C. Brcchler V, Kcrvran A, Blache P, Le-Nguyen Do Laurent S, Bataille D, Pecker F (1991) Miniglucagon [glucagon-(19-29)] is a component of the positive inotropic effect of glucagon. Am J Physio1260, C993-C999 5 Bunnett NW (1987) Post-secretory metabolism of peptides. Am Rev Resp Dis 136. $27-$34 6 Tatemoto K, Carlquist M, Mutt V (1982) Neuropeptide Y: a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nauere (London) 296, 659-660 7 Laburthe M (1990) Peptide YY and neuropeptide Y in the gut. Trends Endocrinol Metab !, 168-174 8 Eberlein GA, Eysselein VE, Schaeffer M, Layer P, Grandt D, Goebell H, Niebel W, Davis M, Lee TD, Shively JE, Reeve Jr JR (1989) A new molecular form of PYY: structural characterization of human PYY (3-36) and PYY (i-36). Peptides 10, 797-803 9 Grandt D. Teyssen S, Schimiczek M, Reeve Jr JR, Feth F, Rascher W, Hirche H, Singer MV, Layer P, Goebell H, Ha FJ, Eysellein VE (1992) Novel generation of hormone receptor specificity by amino terminal processing of peptide YY. Biochem Biop~,s Res Commun 186, 1299-1306 10 Gee NS. Matsas R, Kenny AJ (1983) A monoclonal antibody to endopeptidase-24.1 !. Its application to immunoadsorbent purification and immuno-

11 12

13

14

15

16

17

18

!9

fluorescent microscopy of kidney and intestine. Biochem J 214, 377386 Booth AG, Kenny AJ (1974) A rapid method for the preparation of microvilli from rabbit kidney. Biochem J 142, 575-581 Kessler M, Acuto O, Storelli C, Muter H, MOiler M, Semenza G (1978) A modified procedure for the rapid preparation of efficiently transporting yes ~les from small intestinal brush border membranes. Their use in investigatin~ some properties of D-glucose and choline transport systems. Biochim Biophys Acta 506, 136-154 Smith PK, Krohn RI. Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NH, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem ! 50, 76-85 Kenny AJ, Booth AG, George SG, Ingrain J, Kershaw D, Wood EJ, Young AT (1976) Dipeptidyl peptidase IV, a kidney brush border serine peptidase. Biochem J 157, 169-182 Abbs MT, Kenny AJ (1983) Proteins of the kidney microvillar membrane: analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Clin Sci 65, 55 i-559 Llorens-Cortes C, Huang H, Vicart P, Gasc JM, Paulin D, Corvol P (1992) Identification and characterization of neutral endopeptidase in endothelial cells from ~,enousor arterial origins. J Biol Chem 267, 14012-14018 Mentlein R, Dahms P, Grandt D, Kruger R (1993) Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regui Pept 49, 133-144 Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yakazi Y, Goto K, Masaki T (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (Land) 332, 497-498 Turner At (1993) Endothelin-converting enzymes and other families of metalloendopeptidases. Biochem Sac Trans 21,697-701