Poul Erik H. Jensen

Lebetase, an α(β)-fibrin(ogen)olytic metalloproteinase of Vipera lebetina snake venom, is inhibited by human α-macroglobulins

Abstract The effects of the plasma proteinase inhibitors α2-macroglobulin (α2M) and the α2M-related pregnancy zone protein (PZP) were evaluated towards the metalloproteinase lebetase, isolated from Vipera lebetina venom. We demonstrate that lebetase interacts with both inhibitors. Cleavage of α2M by lebetase resulted in the formation of 90-kDa fragments, and covalent complexes of α2M with lebetase were observed. The proteolytic activity of lebetase against fibrinogen and azocasein could be inhibited by α2M. Cleavage of PZP also resulted in the formation of 90-kDa fragments, and complexes of both dimer and tetramer forms of PZP with lebetase were detected. The amino acid sequence identification of the sites of specific proteolysis of α2M and PZP demonstrate that the cleavage sites are within the bait regions of both proteins. Lebetase I cleaves between Arg696–Leu697, which is one of the most common cleavage sites in α2M by proteinases. The other two cleavage sites in α2M by lebetase are Gly679–Leu680 and His694–Ala695. The cleavage between Pro689–Gln690 is the only cleavage site identified in PZP. In that lebetase is an anticoagulation agent in vivo, we propose that the interaction of lebetase with α2M may suggest a reduced fibrin(ogen)olytic activity of lebetase in human.

Use of hydrophobic affinity partitioning as a method for studying various conformational states of the human α-macroglobulins

The serum proteins alpha 2-macroglobulin and pregnancy zone protein undergo major conformational changes when complexed with proteinases. It is shown that the changes in delta log Kmax determined by hydrophobic affinity partitioning is a measure of the extent of changes in the conformation of these alpha-macroglobulins. We introduce a new term for the changes of surface hydrophobicity in a protein as delta log Kacc. This defines the difference of delta log Kmax between a modified and an unmodified conformational state of a specific protein and can be useful as a parameter to describe the apparent conformational changes in the protein.

Preparation and characterization of a C-terminal fragment of pregnancy zone protein corresponding to the receptor-binding peptide from human α2-macroglobulin

Abstract Digestion of the pregnancy zone protein with papain at pH 4.5 yields an 18 kDa C-terminal fragment. This fragment consists of the 145 C-terminal amino-acid residues cleaved at Asn- 1288 Ile and is homologous to the C-terminal receptor binding fragment of human α 2 -macroglobulin obtained by cleavage with papain. The fragment contains an intrachain disulfide bond between 1308 Cys and 1423 Cys corresponding to that between 1304 Cys and 1419 Cys in α 2 -macroglobulin. An oligosaccharide chain, is present in the C-terminal fragment of pregnancy zone protein as in human α 2 -macroglobulin. The PZP C-terminal fragment was demonstrated to bind to the LRP/ α 2 M-receptor. Both the pregnancy zone protein and α 2 -macroglobulin fragments bind three mAbs ( α 1:1, R35, and 7H11D6) generated against α 2 -macroglobulin. The mAb 7H11D6 was generated against the α 2 -macroglobulin-proteinase complex (Isaacs, I.J., Steiner, J.P., Roche, P.A., Pizzo, S.V. and Strickland, D.K. (1988) J. Biol. Chem. 263, 6709–6714) and the binding of this to the C-terminal fragments of both pregnancy zone protein and α 2 -macroglobulin indicates that both proteins use the same receptor recognition site for binding to the LRP/ α 2 M-receptor.

Proteinases from the fibrinolytic and coagulation systems: Analyses of binding to pregnancy zone protein, a pregnancy-associated plasma proteinase inhibitor

Summary The inhibitory effects of pregnancy zone protein (PZP) on proteinases within the fibrinolytic and coagulation systems have been studied and compared to that of human α2-macroglobulin (α2-M). Plasmin, t-PA, urokinase, thrombin, human plasma kallikrein, human and porcine tissue kallikrein were tested for binding to PZP and α2-M. PZP was cleaved at the ‘bait’ region as seen in SDS-PAGE, by both human and porcine tissue kallikrein, but not by any of the other proteinases tested and we therefore suggest that PZP may have a role in inhibition of tissue kallikrein. Plasmin, thrombin, and plasma kallikrein were found to be bound and inhibited by α2-M by cleavage of the ‘bait’ regions. Minor amounts of cleavage products of α2-M were detected with t-PA and urokinase after prolonged incubation at room temperature. Cleavage of α2-M was detected following incubation with porcine tissue kallikrein, but no cleavage was seen following incubation with human tissue kallikrein. The fast inhibition of plasmin, thrombin, and plasma kallikrein suggest that α2-M may be physiological relevant as an inhibitor for these proteinases. Despite the similarities between α2-M and PZP, significant differences are observed in the inhibition of proteinases. These results suggest distinctive differences in the function of the two human α-macroglobulins. PZP does only seem to inhibit human tissue kallikrein of the proteinases tested from the fibrinolytic and coagulation systems.

Disulfide Bond Formation by Methanethiolation of the Thiol Ester Sulfhydryl Group of α2M

Modification of human a2M by methylamine changes the conformation of native azM drastically by reacting with the glutamyl residues of thiol esters. The conformational changes include closure of the “trap” and exposure of the receptor-recognition sites. Simultaneous treatment of azM with methyl methanethiosulfonate (MMTS) inhibits the conversion of a2M with methylamine and generates a complex with similar structure as native a2M. This is demonstrated by the “slow” electrophoretic mobility of the complex in nondenaturing PAGE and by the ability of the derivative to generate complexes with chymotrypsin.’ Furthermore, the methylamine and MMTS-modified a2M is not removed from circulation in mice as is the methylamine-treated form, but treatment with chymotrypsin converts the derivative to a form removable from circulation in vivo. The methylamine and MMTS-modified derivative has no free titratable thiol groups (TABLE 1) and the methanethiolated bonds are stable for several hours (FIGURE 1). These are also stable after bait region cleavage with chymotrypsin. Even though the methanethiolation is stable, the “slow” electrophoretic form decays to a “fast” form with a half-life of 9 hours. This open “trap” structure with the thiol esters

Binding of Lipoprotein Lipase to α2‐Macroglobulin

Human a*-macroglobulin (a2M) is a large plasma glycoprotein (720 m a ) . The protein consists of four identical subunits covalently held together as dimers and furthermore stabilized as tetramers by noncovalent forces. a2M is a unique proteinase inhibitor able to inhibit proteinases from all four major classes of proteinases. After proteolytic cleavage, a conformational change occurs that allows the proteinase to covalently bind to a2M. It has been demonstrated in vitro that nonproteolytic proteins, present during the proteolytic activation of a2M, can become covalently bound to a2M, for example, insulin. Recent investigations have demonstrated that alM can also bind a number of growth factors and cytok

Cloning and Expression of the 15-kDa C-Terminal Peptide from the Human α2-Macroglobulin as a Fusion-Protein Product in a Prokaryotic Cell Line

es.’ The a2M-proteinase complex is specifically cleared from the circulation by receptor-mediated endocytos i~ .~~~ Much interest has been focused on the a2M receptor because it has been demonstrated that this receptor is identical to the low density lipoprotein receptor-related protein (LRP). Besides a2M-proteinase complexes, the receptor can bind apolipoprotein E-containing lipoproteins: and this binding is enhanced by lipoprotein lipase (LPL): which is shown to bind to the receptor as we11.6 LPL is a 110-kDa homodimeric glycoprotein that interacts with lipids, heparin, and apoprotein CII. LPL is a key enzyme in the catabolism of triglyceride-rich lipoproteins.’ During studies of the binding of LPL to lipoproteins by incubation of bovine 1251labeled LPL (I2%bLPL) with plasma followed by nondenaturing electrophoresis for separation of lipoprotein particles, we observed a radiolabeled band with a Stokes diameter similar to that of a2M.8 The purpose of the present study was to analyze the putative binding of bLPL to aZM. In vitro experiments were therefore performed to study the binding of purified ‘Z51-labeled bLPL9 to purified a2M.’ FIGURE 1 shows the in vitro binding of lZ51-bLPL to azM. FIGURE 1A shows the mobility in a polyacrylamide gel of native a2M as well as different derivatives of azM after incubation with LPL. Native a2M (lane 1) has a “slow” mobility according to the open “trap” structure of the molecule. Upon treatment with methylamine, the “trap” is closed and the mobility becomes “fast” (lane 2). Treatment of azM with chymotrypsin also leads to a “fast” mobility (lane 3). FIGURE 1B shows the autoradiography of the gel in FIGURE 1A. The 12%bLPL bound to native azM (lane l), but not to any of the “fast”

Differences in the proteinase inhibition mechanism of human α2-macroglobulin and pregnancy zone protein

A cDNA fragment encoding the 18-kDa C-terminal peptide from human az-macroglobulin (Ha,M) was amplified, by a modification of the “Sticky Feet” polymerase chain reaction (PCR) protocol,’ from the full-length Ha2M cDNA insert in mammalian expression vector plasmid p l 167.2 To that effect, published nucleotide sequences of the Ha2M gene3 and pACA4 were studied to design chimeric oligonucleotide primer pairs: SFFA2-M and SFRA2-M, each incorporating nucleotide sequences complementary on their 5‘ stretch to the vector plasmid pACA and on their 3‘ stretch to the gene encoding the 18-kDa C-terminal peptide from Ha2M. Besides being phosphorylated on the 5‘ end, the forward primer incorporated nucleotide codons coding for Gly-GlyAsn-Gly residues at the chimeric junction to facilitate site-specific cleavage by hydroxylamine. The “Sticky Feet” PCR product was annealed to and inserted into the uracil-containing single-strand vector plasmid pACA having a strong T7 promoter preceding the gene for wild-type human carbonic anhydrase (HCAII) and was repaired upon transformation into E. coli (TGl), generating pazMCA (diagrammatic representation of the cloning strategy as depicted in FIGURE 1). The 18-kDa C-terminal peptide from HaZM was expressed as fused to the C-terminus of HCAII in E. coli (BL21/ DE3). The expression of the fusion-protein was established by SDS-PAGE (FIGURE 2) and Western blot techniques employing monoclonal antibodies (mAb) that were raised against the 18-kDa C-terminal peptide from Ha2M procured by papain digestion of the methylamine-treated Ha2M molecule. Because an earlier attempt to express Ha2M-RBD (RBD = receptor binding domain) resulted in insoluble product5 and, in our hands, in a low-yield peptide highly susceptible to proteolytic degradation, adoption of this novel approach would enable the generation of the unglycosylated 15kDa C-terminal peptide from Ha2M fused to the C-terminus of the 30-kDa HCAII, thereby protecting against proteolytic degradation and facilitating renaturation from inclusion bodies. The inclusion bodies have been solubilized in 5 M guanidine