Kallikreins, kinins and growth factor biosynthesis

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Reviews Kailikreins, kinins and growth factor biosynthesis

Nerve growth factor biosynthesis Nerve growth factor (NGF) is synthesized as a 33.8 kDa precursor containing a signal peptide of approximately 25 amino acids, and four pairs of basic residues which represent potenCatherine C. Drinkwater, Bronwyn A. Evans and tial cleavage sites 8. The only interRobert I. Richards mediate which has been observed experimentally is the 22 kDa form resulting from the cleavage at Argl15The process of cleaving one protein with another is a very basic one in biology. Arg116 (Ref. 6) (Fig. la). Incubation of In many organisms mature proteins are released from their precursor forms by the isolated submandibular glands with action of proteases. Proteolysis is used extensively, in other important processes such [35S]cysteine demonstrates accumulaas the coagulation cascades and in the biosynthesis of biologically active peptides. tion of the 22 kDa form for one hour, Peptide biosynthesis is characterized by the limited, specific digestion of substrates with a slower but steady increase in at a variety of types of cleavage sites. Analysis ~f the genes coding for glandular 13.2 kDa NGF. The smaller moiety kallikreins, a group of presumptive prohormone' processing enzymes, has enabled represents the 118 amino acid peptide elucidation of the complete structure of the enr.ymes and determination of sites required for neurite outgrowth in vitro of gene expression. The physical properties of these enzymes and their interaction and the development of sympathetic with their substrates provide a unique opportunity to analyse structure-function and sensory neurones in vivo 8. This relationships. mature form of NGF is produced by cleavage at Lys186-Arg187, and at the Arg305-Arg306 pair one residue upSuperficially, the process of synthesis whose involvement in a-factor biosyn- stream from the C-terminus of the preof a high molecular weight protein pre- thesis has been genetically defined 3. cursor. Nerve growth factor isolated from cursor and its limited, specific proteoly- Multicellular organisms would appear sis to an active peptide would appear to to make use of a variety of types of the salivary gland of adult male mice is be a waste of energy and biosynthetic enzyme,, for example, the aspartic acid found as part of a high molecular capacity. Yet this theme is repeated for protease renin cleaves angiotensi- weight complex consisting of two molemost peptide hormones and growth nogen, while the serine protease kalli- cules of mature NGF, denoted the factors in single-celled eukaryotes and krein cleaves kininogen 4. A single gene 13-subunit, 2 ct-subunits, 2 ~/-subunits multicellular organisms alike. In spe- encodes renin in most species while and two atoms of Zn 2+ (Ref. 9, and cific instances the precursor molecule kailikrein is a member of a multigene references cited therein). The additional finding that the 7-subunit is may provide alternative products. For family. capable of cleaving the 22 kDa form to The number and variety of prohorexample the protein pro-opiomelanocortin is the precursor to a number of mones and therefore cleavage sites give mature NGF in vitro provides active peptides and the products of pro- have led many investigators to suggest strong evidence that this protease is cessing differ in each of two cell types the existence of families of processing responsible for the final cleavage expressing the gene 1. Other precursors enzymes capable of the required func- events in vivo 6. The ct-subunit, which is have multifunctional domains which tional diversity 5. The mouse glandular also a member of the glandular kailiappear to act at different sites in the kallikrein gene family has therefore krein family, has no proteolytic activity one physiological process, (e.g. the been a prime candidate for a related due to a series of small deletions in the multiple roles of kininogen in inflam- group of prohormone processing en- coding region of the gene ~°. It has been zymes, meeting the above criteria. In suggested that formation of the high mation2). The processing of these peptide pre- addition to the kinin-generating kalli- molecular weight complex is necessary cursors is of particular interest as the krein found in the kidney, pancreas and for stabilization of NGF, since the responsible enzyme(s) often need to be salivary gland, several members of the ~-subunits alone rapidly lose 90% of coordinately expressed with their sub- mouse glandular kallikrein gene family their biological activity (see references strate and in certain cases have suffici- have been implicated in the synthesis of cited in Ref. 9). This stabilization may ent specificity to cleave at selected sites biologically active peptides. For exam- be particularly important within the on selected protein molecules. Several ple, detailed studies on the biosynthesis secretory vesicles of cells producing classes of protease have been proposed of nerve growth factor (NGF) 6 and NGF. Although NGF has been identified in as processing enzymes yet the evidence epidermal growth factor (EGF) 7 in for only a few of these is clear cut. The mouse salivary gland have demon- a variety of mammalian tissues, includbest example is the yeast kex2 protease strated the involvement of the kalli- ing regions of the brain H, it has not kreins ~/-NGF and EGF-binding been possible so far to detect expresc. c. Drinkwater,B. A. EvansandR. L Richards protein respectively (Fig. 1). In each sion of the a- and ~,-subunits in any are at the Howard Florey Institute of Experi- case the kallikrein remains bound to its tissue other than mouse salivary gland mental Physiology and Medicine, University of (B. A. Evans, unpublished). Given Melbourne, Royal Parade, Parkville, Victoria, substrate after cleavage, forming a that the amounts of NGF found in stable high molecular weight complex. 3052,Australia. c~ 1988, ElsevierPublicationsCambridge 0376-5067/88/$02.00

170

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pro NGF (33.8kDa) pre

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proNGF (22kDa)

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. s N G F ' ~ 7S NGF COMPLEX

(13.2 kDa)

~

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1

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Kallikrein

Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg (¢)

Lys-bradykinin

other tissues are orders of magnitude lower than those in the salivary gland, however, it is possible that the levels of the a- and 7-subunits or the corresponding mRNAs are below the limits of detection of the procedures used. The biosynthesis of active NGF is further complicated by the recent finding that differential splicing of proNGF mRNA leads to the formation of two different precursor proteins~2.The longer form is predominant in mouse submandibular gland, as well as certain myoblast cell lines, whereas the short form is present in mouse placenta, embryonic hindlimb and mouse L cells, a fibroblast cell line ~2. The two forms of NGF precursor differ in the region likely to be used as a signal peptide, and may well be directed to different parts of the cell during translation. This observation is of interest in relation to L cell NGF, since this peptide is associated with proteins distinct from the aand 7-subunits found in the salivary gland 13. It is possible that NGF arising from the shorter precursor form is activated via a different processing pathay from that in tissues expressing the long precursor.

Epidermal growth factor biosynthesis Mouse epidermal growth factor (EGF) is a 53 amino acid protein (6 kDa) with potent mitogenic effects ~4. Surprisingly, given the small size of mature EGF, its mRNA encodes a 1217 amino acid protein ~s. This appears to be processed by a series of steps, through at least two interme-

Fig. 1. Biosynthesis of (a) nerve growth factor (NGF) (b) epidermal growth factor (EGF) and (c) kallidin (Lys-bradykinin). In each case the location of the mature product (hatched) within the precursor protein is shown. Both ~I-NGF and EGF-BP remain bound to their respective growth factors through the carboxyterminal arginine, whilst renal kallikrein dissociates from Lys-bradykinin after cleavage. The exact positioning of a-NGF within the 7S NG F complex is not known.

Expression of mouse preproEGF in salivary glands exhibits sexual dimorphism, with levels in males being an order of magnitude higher than in females 2°. All three EGF-BPs are also expressed in higher quantities in male salivary glands than in female salivary glandslg, producing corresponding sexual dimorphism in levels of the mature protein 14. PreproEGF expression has also been detected in mouse kidney 2°. However, whilst antibodies to mouse preproEGF precipitate mature 6 kDa EGF from mouse salivary glands, they only precipitate the 130 kDa precursor protein from kidney 2°. It is consistent with an essential role in EGF processing that none of the EGF-BPs are expressed in the kidney 19.

Renal kallikrein (kininogenase) and kinin generation The hypotensive activity of kallikrein was demonstrated to be a result of its proteolytic action on high and low diates (125 kDa and 110 kDa), to a 9 molecular weight kininogen to release kDa EGF precursor 16. Finally, a kallidin, or Lys--bradykinin, a peptide carboxy-terminal fragment is removed with contractile and vasoactive by EGF-binding protein (EGF-BP), properties. As a consequence, renal another member of the gland,;ar kalli- kallikrein is sometimes referred to as krein family, to produce the mature kininogenase (see Ref. 4 and refergrowth factor 7. In an analogous man- ences cited therein). ner to high molecular weight NGF, In many respects the kininEGF-BP remains bound to EGF generating member of the kallikrein through the arginine at its new carboxyl gene family appears to be an exception. terminus ~. The high molecular weight Rather than being coordinately EGF complex of 74 kDa consists of two expressed in the liver with its substrate molecules each of substrate and kininogen, it is in general expressed at enzyme. The formation of this complex the site of action of the active cleavage is again thought to provide a means for product, kinin. The major sites of stable storage of EGF in the secretory kininogenase expression are the granules. salivary glands, pancreas and kidney, At first there was some confusion as with expression also detected in the to the identity of EGF-BP. One pituitary, and the gut (Ref. 4 and refergroup 17 had sequenced two proteins, ences cited therein). This relationship EGF-BP type A and type B, from the between expression and site of action high molecular weight complex, whilst would be consistent with kallikrein another group TMhad isolated only one having an important regulatory funcdifferent protease (EGF-BP type C). tion in kinin generation. The de novo All three proteins are clearly members synthesis and release of the enzyme of the kallikrein family 19. Analysis of appears to be critical since the substrate the respective genes demonstrated that (kininogen) circulates in abundance, all three proteins are encoded by the the enzyme activity can be readily inhione strain of mouse and therefore do bited by circulating serine protease not represent strain polymorphisms or inhibitors, and the locally produced alleles ~9. Furthermore, it became kinin is rapidly degraded by kininases, obvious from comparison of the com- such as converting enzyme. plete structure of all three enzymes that the amino acid sequence data con- Substrate specificity of glandular tained residues from all three pro- kallikreins teins ~7'19. Only one of the enzymes, The substrate specificity of individEGF-BP type C, has been shown to be ual members of the mouse kallikrein able to cleave the 9 kDa EGF precursor family is very high. Kininogens of some and reconstitute the high molecular species are often poor substrates for the weight complex TM. kallikreins of others. Though 7-NGF

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(a) Mouse glandular kallikreins

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DT-- G - S SWGY--G-DT--G-S SWGH-- G - YT--H-S SWGP--S-DT--G-S SWGP-- G--DT-- G-S SWGP-- G-NI- G-S--STGP --A-DT-- G - S SWGF--G-AWGP-- G-DT-- D - S SYGP--A-GP-- G - S SIGP--A--SWGS--A-DT-- G - S SWGS--.A--

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Fig. 2. (a) Comparison o f mouse glandular kallikreins. The depicted amino acid residues (given in one letter code) correspond to those in porcine pancreatic kal!ikrein that directly interact with bovine trypsin inhibitor s2, and may therefore be involved in determining substrate specificity. Dashed lines indicate incomplete sequence data. The amino acid sequences are derived from nucleotide sequences: m G K - I zS, m G K - 3 and ¥o, mGK_SZS, mGK..6z6, mGK..827, mGK-9, 13 and 2219, mGK-II, 16 and 213J (C. C. Drinkwater, unpublished). (b) The corresponding amino acid residues from the kinin-generating protease (renal kallikrein) o f mouse z6, rat~° pig 31 and human 29 are also depicted. Numbering is relative to the amino-terminal isoleucine (llel ) o f mature mouse 7-NG F.

and EGF-BP do exhibit some kininogenase activity, this is one to two orders of magnitude less than that of renal kallikreineL General protease activity of several kallikreins was compared with that of trypsin, and found to be considerably less, indicating stringent substrate requirements2L Addition of 7-NGF to proEGF had no effect, even though molar ratios of up to 500:1 were used 7. Likewise, EGF-BP was shown to be unable to substitute for 7-NGF to reconstitute 7S NGF 7. Only one member of the mouse kallikrein family, 7-renin, cleaves synthetic renin substrate e2. This specificity extends to enzymes from other species, with rat tonin specifically cleaving renin substrate 23 and human prostate antigen selectively cleaving high molecular weight SV protein from seminal vesicles24. Comparison of the amino acid sequer,ces of these respective enzymes reveal:~ residues likely to be important for substrate specificity. The structure of the .functional mouse genes has now been determined mz5-28 (C. C. Drinkwater, unpublished) as has the structure of renal kall~krein from several species z°,29-31. Figure 2 shows a comparison of these sequences highlighting those residues thought to be involved in determining specificity. Crystallization of porcine pancreatic kallikrein (both on its own and corn-

plexed with bovine trypsin inhibitor) indicates residues that may be important in substrate recognition and catalysis32. The two most notable are Tyr93* and Trp205 which form an empty wedge-shaped hydrophobic gap, thus promoting binding of phenylalaninecontaining peptides 32. Renal kallikreins from all species have these two residues, though they are not conserved in all mouse glandular kallikreins (Fig. 2). Similarly each of the kailikreins of known specificity has unique amino acids at positions likely to determine specificity ~°'t9'26. Accordingly, these residues provide obvious targets to address the iss-e of enzymesubstrate determinants. Kallikrein gene families in different species Renal kallikrein has,been found in all mammalian species examined. In the mouse and human genomes, this enzyme is encoded by a single gene which is also expressed in the pancreas and salivary gland 26'e9. Differences between the species arise when we consider the number and expression of other members of the gene family. We have found an additional 11 functional *Amino acid residues are identified by numbers corresponding to the homologous amino acids of mouse 7-NGF °, relative to the amino-terminal isoleucine (lle l ) o f the mature protein.

genes in the mouse, all of which are expressed in testosterone-responsive secretory cells of the male salivary g l a n d 33"34, (C. C. Drinkwater, unpublished). Among the products of these genes are the 7-subunit of NGF and EGF-BP. The human genome, on the other hand, appears to have only two additional kallikrein-like genes. One encodes prostate-specific antigen 35,, while expression of the other, hKK3, has not been detected 29. In all species examined expression of functional genes other than that coding for the kinin-generating enzyme (renal kallikrein) seems to occur only in testosterone-responsive cell types such as those in the salivary and prostate glands. Examination of the kallikrein families in different species clearly has implications for questions such as the activation of human NGF and EGF, since there are no equivalent human genes to those coding for mouse 7-NGF and EGF-BP. A trivial explanation might be that the involvement of these enzymes in mouse growth factor biosynthesis is an enigmatic property of this organism's unusual salivary glands. No other species produces such an enormous amount of these growth factors in this (or any other) tissue. An alternative explanation is that the mouse enzymes are unique in their ability to remain bound to their substrate after cleavage, necessitating a greater variety (and number) of enzymes. It is possible that in other species the cleavage event is truly catalytic, with one or a few enzymes able to cleave a wide range of substrates with the required specificity. The differences between species suggest that unifying themes in peptide biosynthesis might be hard to come by, with only a few crucial examples, such as the relationship between kininogenase and its substrate, conserved through evolution. References Limited space has prevented citation of some of the primary literature which can be found in the articles listed below. 1 Eipper, R. A. and Mains, R. E. (1980) Endocrine Rev. 1,1-27 2 Mfiller-Esteri, W., lwanaga, S. and Nakanishi, S. (1986) Trends Bioehem. ScL 11, 336-339 3 Julius, D., Brake, A., Blair, L., Kunisawa, R. andThorner, J. (1984) CeU37,1075--1089 4 Schachter, M., Longridge, D. J., Wheeler, G. D., Mehta, J. G. and Uchida, Y. (1986) J. Histochem. Cytochem. 34,927-934 5 Marx, J. (1987) Science 235,285-286 6 Berger, E. A. and Shooter, E. M. (1977) Proc. Natl Acad. ScL USA 74, 3647-3651

TIBS 13- May 1988

172 7 Frey, P., Forand, R., Maciag, T. and Shooter, E. M. (1979) Proc. Natl Acad. Sci. USA 76, 6294--6298 8 Scott, J., Selby, M., Urdea, M., Quiroga, M., Bell, G. I. and Rutter, W. J. (1983) Nature 302, 538~540 9 Thomas, K. A., Silverman, R. E., Jeng, I., Baglan, N. C. and Bradshaw, R. A. (1981) J. Biol. Chem. 256, 9147-9166 10 Evans, B. A. and Richards, R. I. (1985) EMBO J. 4,133-138 11 Korsching, S., Auburger, G., Heumann, R., Scott, J. and Thoenen, H. (1985) EMBOJ. 4, 1389-1393 12 Selby, M. J., Edwards, R., Sharp, F. and Rutter, W. J. (1987) Mol. Cell. Biol. 7, 30573064 13 Pantazis, N. J. (1983) Biochemistry 22, 42644271 14 Carpenter, G. and Cohen, S. (1979) Annu. Rev. Biochem. 48,193-216 15 Gray, A., Dull, T. J. and Ullrich, A. (1983) Nature 303,722-725 16 Burmeister, M., Avivi, A., Schlessinger, J. and Soreq, H. (1984) EMBOJ. 3,1499--1505 17 Anundi, H., Ronne, H., Peterson, P. A. and Rask, L. (1982) Eur. J. Biochem. 129, 365371 18 Isackson, P. J., Siiverman, R. E., Blaber, M., Server, A. C., Nichols, R. A., Shooter, E. M.

and Bradshaw, R. A. (1987) Biochemistry 26, 2082-2085 19 Dri~.kgeter, C. C., Evans, B. A. and Richards, R. I (~987) Biochemistry 26, 67506756 20 Rail, L. B., Scott, J., Bell, G. I., Crawford, R. J., Penschow, J. D., Niall, H. D. and Coghlan, J. P. (1985) Nature313, 228-231 21 Bothwell, M. A., Wilson, W. H. ~nd Shooter, E. M. (1979) J. Biol. Chem. 254, 7287-7294 22 Poe, M., Wu, J. K., Florance, J. R., Rodkey, J. A., Bennett, C. D. and Hoogsteen, K. (1983) J. Biol. Chem. 258, 2209-2216 23 Lazure, C., Seidah, N. G., Thibauit, G., Boucher, R., Genest, J. and Chretien, M. (1981) Nature 292,383-384 24 Lilja, H. (1985)J. Clin. Invest. 76,1899-1903 25 Mason, A. J., Evans, B. A., Cox, D. R., Shine, J. and Richards, R. I. (1983) Nature 303,300-307 26 van Leeuwen, B. H., Evans, B. A., Tregear, G. W. and Richards, R. I. (1986) J. Biol. Chem. 261,5529-5535 27 Fahnestock, M., Brundage, S. and Shooter, E. M. (1986) Nucleic Acids Res. 14, 48234835 28 Drinkwater, C. C. and Richards, R. I. (1987) Nucleic Acids Res. 15,10052 29 Evans, B. A., Zhang, X. Y., Close, J. A., Tregear, G. W., Kitamura, N., Nakanishi, S.,

Organization of lipids in the plasma membranes of malignant and stimulated cells: a new model Carolyn E. Mounfford and Lesley C. Wright Neutral lipids make up about 6% of the lipid content of plasma membranes from malignant cells. Magnetic resonance spectroscopy (MRS) identifies this neutral lipid as predominantly triglyceride which is not in bilayer form. A new structural model is proposed whereby neutral lipid domains are intercalated with the bilayer lipid of the plasma membrane. A functional role for these neutral lipid domains is also proposed based on plasma membrane alterations which occur with cellular stimulation, with the acquisition of resistance to anti-cancer drugs, and in metastatic cells. The fluid mosaic membrane model ~, proposed to account for the packing of phospholipids in a membrane bilayer, has proved difficult to verify whilst the membrane remained part of intact cells. The major difficulty was that the length of time required to obtain the data far exceeded the time the cells remained viable. Consequently, physical methods able to ascertain if lipids were in the bilayer configuration have C. E. Mount ford and L. C. Wright are at the Ludwig Institute for Cancer Research (Sydney Branch) and Centenary Institute for Cancer Medicine and Cell Biology, Blackburn Building, University of Sydney, Sydney, NSW 2006, Australia.

© 1988.ElsevierPublicationsCambridge 0376-5067/a8/$02.00

been mostly restricted to model membranes. Fluorescence polarization methods are more conducive to the study of intact cells but require a probe molecule, the ultimate location of which is always contentious as is the effect of the probe itself on the membrane2. To further complicate matters no information was available on the neutral lipid composition of highly purified plasma membranes since it had been assumed that triglycerides and cholesteryl esters were not membrane components. Magnetic resonance spectroscopy (MRS) is one of the few techniques that permits the direct observation of tool-

CaUen, F., Baker, E., Hyland, V. J., Sutherland, G. R. and Richards, R. I. Biochemistry (in press) 30 Swift~ G. H., Dagorn, J-C., Ashley, P. L., Cummings, S. W. and MacDonald, R. J. (1982) Proc. Natl Acad. Sci. USA 79, 7263-7267 31 Tschesche, H. et al. (1979) in Kinins I1 Biochemistry, Pathophysiology and Clinical Aspects. Advances in Experimental Medicine and Biology, Vol. 120A (Fujii, S., Moriya, H. and Suzuki, T., eds), pp.245-260, Plenum Press 32 Bode, W., Chen, Z., Barteis, K., Kutzbach, C., Schmidt-Kastner, G. and Bartunik, H. (1983)J. Mol. Biol. 164,237-311 33 Evans, B.A., Drinkwater, C. C. and Richards, R. I. (1987)J. Biol. Chem. 262, 8027-8034 34 van Leeuwen, B. H., Penschow, J. D., Coghlan, J. P. and Richards, R. I. (1987) EMBO J. 6,1705-1713 35 Watt, K. W. K., Lee, P. J., M'Timkulu, T., Chan, W. P. and Loor, R. (1986) Proc. Natl Acad. Sci. USA 83, 3166-3170

A review by J. Thorner on the yeast kex2 protease is planned for a future issue of TIB$

ecules in intact cells. Small molecules usually generate a narrow-lined spectrum due to a high degree of molecular freedom, and protons in different chemical environments can be resolved in a spectral width of 10 ppm. In contrast, large molecules and solids yield broad spectra. Although there is some residual motion of phospholipids in the bilayer structures of membranes their spectra are of the order of 40 ppm spectral width 3,4. A combination of chemical data from leukaemic lymphoblast membranes purified 45- to 600-fold by ultra-centrifugation methods, and twodimensional MRS methods have allowed us to propose a new structure and function for plasma membranes of malignant, embryonic and stimulated cells. Furthermore the development of a new rapid method for the simultaneous acquisition of broadline and high resolution spectra from cells s supports the notion that some, but not all, membrane lipid is in the bilayer configuration6. Magnetic resonance spectroscopy of membranes and cells The high resolution proton ( I H ) MR spectrum obtained from malignant, embryonic and stimulated cells is that of lipid7 and represents the highly mobile lipids contained by these cells. These lipids were thought unlikely to be part of the bilayer since lipid in this