Cell surface peptidases are neither peptide- nor organ-specific

40

TIBS 11 - January 1986

Cell surface peptidases are neither peptide- nor organ-specific John Kenny The temptation to name a peptidase activity after the peptide which happened to be the starting point of the research is understandable, but usually misleading. The surface of kidney microvilli is covered with a battery of peptidases which provide an excellent reference collection with which to compare peptidase activities detected on other cell surfaces. One such example is endopeptidase-24.11, the wide distribution of which extends to some regions of the central nervous system, where it may have an important role in the inactivation of neuropeptide transmitters. Brush border membranes are extraordinarily rich in hydrolases; these proteins are ectoenzymes with their bulk, including the active site, exposed at the external surface of the membrane and inserted into the lipid bilayer by a short stalk leading to a hydrophobic domain 1,2. In the kidney, most of the known brush border hydrolases are peptidases, several of which are major constituents of the membrane. This group of enzymes is listed in Table 1. Most of those characterized in detail have been shown to be metaUoenzymes, though dipeptidyl peptidase IV is a serine enzyme. They seem to form an organized system rather than a random collection; in pig (as in human) there is a single endopeptidase, capable of initiating attack on most peptides, supported by a group of exopeptidases able to exploit the smaller peptides thus released. Collectively, they ensure complete hydrolysis of any peptides (but not proteins) filtered by the glomerulus. The exopeptidases attack at either the N- or C-terminus and release either single amino acids or dipeptides. Aminopeptidases predominate over carboxypeptidases; in particular, microvilli seem to lack a broadly specific carboxypeptidase to match aminopeptidase N, but this type of activity has so far eluded the search. In fact, it is likely that the list of enzymes is not yet complete: a very recent addition is aminopeptidase W, an enzyme preferring small peptides with Trp as the second residue. Its discovery was unusual in that it was first isolated by a monoclonal antibody as a protein of unknown function and only later shown to be a new microvillar peptidase 3. The brush border peptidases have been identified and assayed using a variety of synthetic substrates or model pepJ. Kenny is at the MRC Membrane Peptidase Research Group, Department of Biochemistry, University of Leeds, Leeds LS2 9JT, UK.

tides; investigators have usually not been tempted to assign their activity to a single specific natural peptide (peptidyi dipeptidase A, often referred to as 'angiotensin I converting enzyme' or 'ACE' is the only exception). In contrast, studies on other tissues or organs, where the degradation of a defined peptide was the main concern, have led to a profusion of terms such as insulinase, glucagonase, enkephalinase, oxytocinase, substance P degrading enzyme, all of which imply a specificity which is either lacking or unproven. The main point of this review is

to show that many different cell types may express the same peptidase on their external surface, the physiological role of which will vary with the particular location. It follows that a limited number of cell surface peptidases (perhaps a dozen) is adequate to deal with the requirements for hydrolysing biologically active peptides in all tissues and organs. Thus unlike receptor binding of such peptides, which is highly specific, the inactivation by peptidases is relatively nonspecific. Several good examples could be used to support this hypothesis - aminopeptidase N, dipeptidyl peptidase IV, peptidyl dipeptidase A - but I will confine myself to describing one enzyme, endopeptidase-24.11.

Properties of endopeWidase- 24.11 Endopeptidase-24.11 has now been purified from kidney4,5 intestine6, pituitary7 and brains. It is a glycoprotein with subunit M r values in this range 87 00094 000, the variation being attributed to differences in the extent and pattern of glycosylation8. That it is an ectoenzyme can be seen from the electronmicrograph of phospholipid lipsomes into which the purified enzyme has been reconstituted9 (Fig.l). Like the other brush border

Table 1. Pig kidney microvillar peptidases Enzyme

Endopeptidase-24.11 a

Active site

Specificity

Nature of residue

Zn

4)-0-11-0-

Hydrophobic

Aminopeptidase N

Zn

11-0-043-

Many

Aminopeptidase A

Ca

I-0-0-0-

Glu/Asp

Aminopeptidase P

-

041-04)-

Pro

Aminopeptidase W

-

0-1-04)-

Trp

Dipeptidyl peptidase IV

Ser

04M)-0-

Pro/Ala

Carboxypeptidase P

Zn

-04)-I-0

Pro

Peptidyl dipeptidase A

Zn

-0-04141

Many

y-Glutamyl transferase

-

1-0-0-0-

7-Glu

Exopeptidases

aln rat and mouse a second metano-endopeptidase is also present.

Table II. Hydrolysis of three neuropeptides by endopeptidase-24.11 Peptide

Sequence

Specificity constant

kcat/Km, (min -r laM-Z) Substance P

RPLPQQFFGLMa

159

[Leu]enkephalin Luliberin (LHRH)

YGG F L pE H W S Y G L R P G a a

44 1.1

aBonds hydrolysed not fully determined. (Data from Ref. 13.)

~) 1986,ElsevierSciencePublishersB.V.,Amsterdam 0376-5067/86/$02.00

T I B S 11 - January 1986

Fig. 1. Endopeptidase-24.11 reconstitutes in liposomes as a typical ectoenzyme. This negatively stained electron micrograph shows a liposome coated with knobs: the hydrophobic anchor associates with the phospholipid bilayer and is linked to the globular protein by a short (2 nm) junctional peptide (or stalk). The protein appears to be dimeric, especially en face. Bar = 50 nm. (Electron micrograph from Ref. 3.)

a

b

Fig. 2. Colocalization of endopeptidase-24.11 and substance P immunoreactivities in adjacent cryostat sections of nucleus interpeduncularis from pig mid brain. (a) Stained by an immunoperoxidase reaction for endopeptidase-24.11 immunoreactivity; (b) similarly stained for substance P immunoreactivity. Both antigens are distributed throughout the neuropil of the nucleus and both tend to be highly concentrated in certain areas (arrows). (Micrographs from Ref. 22.)

41 hydrolases it is a 'stalked' protein, a homodimer (i.e. two identical subunits) with a highly asymmetric orientation with respect to the lipid bilayer. Its action in vivo is thereby confined to hydrolysis of susceptible substrates at the membrane surface. The general specificity was first determined by using insulin B chain as a substrate; all the products were the result of hydrolysis at bonds involving amino groups of hydrophobic amino acids4 such as Phe, Tyr, Leu, Ile and Val. One group of microbial enzymes (that includes thermolysin) hydrolyses insulin B chain in the same manner; like endopeptidase-24.11 they are zinc-proteins and a potent inhibitor of thermolysin, called phosphoramidon, is an even better inhibitor for endopeptidase-24.11 (Ref. 10). The microbial and mammalian enzymes share the same catalytic mechanism, but are clearly different in respect of structure, immunological determinants, and detailed specificity. The basic specificity of the enzyme relating to a single hydrophobic residue in the substrate is only part of the story. Using a series of synthetic peptide substrates, Marian Orlowski and his colleagues7.11.12 have shown that the mammalian endopeptidase has a rather extended active site in which binding of side chains of substrate amino acids remote (sequentially) from the cleavable bond may influence the efficiency of hydrolysis, Glutaryl-Phe-Gly-Phe-2-naphthylamide has a specificity constant (K~t/Km) nearly 200 times greater than GlutaryI-GlyGly-Phe-2-naphthylamide, Phe being the best residue in the P1' position (where hydrolysis is at the P1-P1' bond). Thermolysin has quite different requirements for subsite binding 12. The manner of binding of amino acid sidechains remote from the point of hydrolysis (e.g. at P2, P3 etc) has a major influence on the efficiency of hydrolysis of neuropeptides. There may be as much as two orders of magnitude difference between specificity constants for the best and worst neuropeptide substrates of endopeptidase 24.1113 (Table II). In effect, studies of this kind have served to highlight those neuropeptides for which the endopeptidase may be important in their metabolism.

Endopeptidase-24.11 is widely distributed but not ubiquitous Two specific reagents have been used to map the distribution of an enzyme which, at first, appeared to be restricted to a renal brush border, later extended to

42

T I B S 11 - January 1986

include intestinal brush borders. The first is the inhibitor, phosphoramidon, which can discriminate in enzymic assays, and the second is a monoclonal antibody, GKTC2, used in a solid phase immunoradiometric assay (i.r.m.a.) TM. Surveys of pig tissues using these methods confirmed that kidney cortex has the highest concentration of endopeptidase-24.11, but revealed high concentrations in lymph nodes, exceeding those in the intestine. Significant amounts were also found in glandular organs such as salivary glands, adrenal glands, pancreas and anterior pituitary. Isolated chondrocytes from articular cartilage were relatively rich in the antigen, which disappeared as the cells dedifferentiated in culture. Crude homogenates of brain and spinal cord were below the threshold of sensitivity of the i.r.m.a. - the antigen could only be detected in membrane fractions 15. Despite these relatively low concentrations in brain, an account of its identification, isolation and detailed mapping in the central nervous system (CNS) will serve to support the main argument of this review.

Endopeptidase-24.11 is responsible for the activity previously known as 'enkephalinase' Crude preparations from brain were known to hydrolyse [Leu]enkephalin at two sites 16 Tyr-Gly-Gly-Phe-Leu The first site is hydrolysed by an aminopeptidase (this can be prevented by using an analogue, Tyr-DAla-GlyPhe-Leu). The second cleavage was thought to be the more important and to be the effect of a specific enzyme, 'enkephalinase'. An inhibitor, thiorphan 17, was synthesized on the basis of the same logical arguments that had led to the synthesis of captopril as an inhibitor of 'ACE '18. Thiorphan was shown to increase the survival of enkephalins in brain slices and possibly to have an analgesic effect in vivo 19. T h e first clue to the identity of 'enkephalinase' came from the demonstration that thiorphan and phosphoramidon were equally good inhibitors of the purified renal endopeptidase-24.11 (Ref. 20). By preparing synaptic membranes from striata dissected from pig brains it was possible to show that the hydrolysis of [DAla2, Leu s] enkephalin and substance P were completely inhibited by phosphoramidon with the same 150 value (8 riM) observed

for the purified renal enzyme. The bonds of substance P hydrolysed by the membranes were exactly those attacked by the purified enzyme21 (Table II). However, immunological evidence was essential to show that the same protein, and not just another enzyme of the same family, was involved in the CNS. A polyclonal antibody was used to inhibit the hydrolysis of enkephalin and substance P by membranes from kidney microviilus and striatal synapse; the titration curves for the inhibition of the activity from both organs were indistinguishable21. The brain enzyme was isolated by immunoaffinity chromatography using the monoclonal antibody GKTC2; it proved to be immunologically and enzymically identical with the renal and intestinal forms 8. The identification of endopeptidase24.11 in the brain does not establish its role. One step towards this goal has been completed recently (Matsas, R., Kenny, A. J. and Turner, A. J., submitted). Our monoclonal antibodies failed to give an adequate signal in cryostat sections of brain tissue, even though they were suitable for immunocytochemistry in many other tissues. The use of a polyclonal antibody has increased the sensitivity of the technique and has enabled the location of the enzyme in the CNS to be mapped in detail. The enzyme is concentrated in certain regions of the brain, especially in the striatum (caudate, putamen and globus pallidus), and in the olfactorv tubercle. In the mid-brain it is concentrated in the substantia nigra and nucleus interpeduncularis. These are peptiderich regions; in particular, substance P has been mapped to the striatal-nigral pathway. Cryostat sections adjacent to those stained for the endopeptidase were immunostained for other antigens. Quite often, strong staining for the enzyme was matched by strong staining for substance P (Fig. 2). Another positive correlation was between staining for endopeptidase and for neurofilament protein (i.e. intermediate filaments specific to neurons). On the other hand, positive matching was not seen for an astrocyte marker glial fibrillary acidic protein. These immunohistochemieal studies suggest that endopeptidase-24.11 is a component of neuronal membranes and that substance P may be one of its natural substrates. But such conclusions are tentative: the details of the location of endopeptidase24.11 requires immunocytochemistry at the electron microscope level and experiments on cultured cells. Proving a role for the enzyme in terminating a peptidergic signal requires extensive physiologi-

cal and pharmacological studies. I have taken the example of one enzyme, endopeptidase-24.11, whose abundance in kidney microvilli was exploited to isolate it in purified form and to generate monoclonal and polyclonal antibodies. These were the essential tools with which the wider role of endopeptidase was revealed. In the brain there is a strong case for its involvement in the inactivation of some neuropeptides. It is likely that it has a similar relationship for some regulatory peptides in the immune and endocrine systems, but these are largely unexplored areas.

References 1 Kenny, A. J. and Maroux, S. (1982) Physiol. Rev. 62, 91-128 2 Semenza, G. Annu. Rev. Cell Biol. 2 (in press) 3 Gee, N. S. and Kenny, A. J. (1985) Biochem. J. 230, 753-764 4 Kerr, M. A. and Kenny, A. J. (1974) Biochem. J. 137,477--488 5 Fulcher, I. S. and Kenny, A. J. (1983) Biochem. J. 211,743-753 6 Fulcher, I. S., Chaplin, M. F. and Kenny, A. J. (1983) Biochem. J. 215,317-323 70rlowski, M. and Wilk, S. (1981) Biochemistry 20, 4942- 4945 8 Relton, J. M., Gee, N. S., Matsas, R., Turner, A. J. and Kenny, A. J. (1983) Biochem. J. 215, 519-523 9 Kenny, A. J., Fulcher, I. S., McGill, K. A. and Kershaw, D. (1983) Biochem. J. 211,755-762 10 Kenny, A.J. (1977) in Proteinases in Mammalian Cells and Tissues (Barrett, A. J. ed.), pp. 393-344, Elsevier/North-Holland Biomedical Press 11 Almenoff, J. and Orlowski, M. (1983) Biochemistry 22, 590-599 12 Pozsgay, M., Michaud, C. and Orlowski, M. (1985) Biochem. Soc. Trans. 13, 44-47 13 Matsas, R., Kenny, A. J. and Turner, A. J. (1984) Biochem. J. 223,433--440 14 Gee, N. S., Bowes, M. A., Buck, P. and Kenny, A. J. (1985) Biochem. J. 228, 119-126 15 Matsas, R., Ranray, M., Kenny, A. J. and Turner, A. J. (1985) Biochem. J. 228,487-,492 16 Schwartz, J.-C., Malfroy, B. and De La Baume, S. (1981) LifeSci. 29, 1715-1740 17 Roques, B. P., Foumir-Zaluski, M. C., Soroca, E., Lecomte, J. M., Malfroy, B., Llorens, C. and Schwartz, J.-C. (1980) Nature Lond. 288,286-288 18 Ondetti, M. A., Rubin, B, and Cushman, W. (1977) Science 196,441-444 19 De Le Baume, S., Yi, C.C., Schwartz, J.C-., Chaillet, P., Marcais-Collado, H. and Constentin, J. (1983) Neuroscience 8, 143-151 20 Fulcher, I. S., Matsas, R., Turner, A. J. and Kenny, A. J. (1982) Biochem. J. 203,519-522 21 Matsas, R., Fulcher, I. S., Kenny, A. J. and Turner, A. J. (1983) Proc. Natl Acad. Sci. U.S.A. 80, 3111-3115 22 Matsas, R., Kenny, A. J. and Turner, A. J. (1986) Neuroscience 18,991-1012