The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.

A lambda gt11 cDNA library containing DNA inserts prepared from human liver mRNA has been screened with an antibody to human alpha 2-thiol proteinase inhibitor that was isolated from fresh plasma. Eighteen positive clones were isolated from one million phage, and each was plaque purified. The cDNA insert of one of these phage was sequenced and shown to code for alpha 2-thiol proteinase inhibitor as identified by a partial amino acid sequence of the light chain of alpha 2-thiol proteinase inhibitor. This cDNA insert contained 1529 base pairs coding for the complete alpha 2-thiol proteinase inhibitor. It included 45 base pairs of 5' noncoding sequence, 1281 base pairs that code for pre alpha 2-thiol proteinase inhibitor, a stop codon, 160 base pairs of 3' noncoding sequence, and 40 base pairs of poly(A) tail. The noncoding sequence on the 3' end contained a potential recognition site (AATAAA) for processing and polyadenylation of precursor messenger RNA. The amino acid sequence of alpha 2-thiol proteinase inhibitor deduced from the cDNA showed a striking similarity (overall homology at 74%) to that of bovine low molecular weight (LMW) kininogen, including two internally repeated sequences and a nonapeptide sequence of bradykinin. These data clearly indicated that alpha 2-thiol proteinase inhibitor and LMW kininogen are identical. This was further supported by immunological cross-reactivity between alpha 2-thiol proteinase inhibitor and LMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Legumain/asparaginyl endopeptidase (EC 3.4.22.34) is a novel cysteine protease that is abundantly expressed in the late endosomes and lysosomes of renal proximal tubular cells. Recently, emerging evidence has indicated that legumain might play an important role in control of extracellular matrix turnover in various pathological conditions such as tumor growth/metastasis and progression of atherosclerosis. We initially found that purified legumain can directly degrade fibronectin, one of the main components of the extracellular matrix, in vitro. Therefore, we examined the effect of legumain on fibronectin degradation in cultured mouse renal proximal tubular cells. Fibronectin processing can be inhibited by chloroquine, an inhibitor of lysosomal degradation, and can be enhanced by the overexpression of legumain, indicating that fibronectin degradation occurs in the presence of legumain in lysosomes from renal proximal tubular cells. Furthermore, in legumain-deficient mice, unilateral ureteral obstruction (UUO)-induced renal interstitial protein accumulation of fibronectin and renal interstitial fibrosis were markedly enhanced. These findings indicate that legumain might have an important role in extracellular matrix remodeling via the degradation of fibronectin in renal proximal tubular cells.

Abstract. An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin . The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces . Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa) . Globulin fractions from normal plasma immunodepleted of high molecu-C ELL attachment and spreading on extracellular matrices are central events in a variety of biological phenomena such as embryogenesis and organogenesis, tumor metastasis, wound healing, and thrombus formation . These events are recapitulated by in vitro assays in which cells attach and spread on protein-coated surfaces. Such assays have given considerable insight about the molecular basis of cell attachment and spreading and led to the identification of the integrin group of heterodimeric adhesion receptors (Hynes, 1987;Ruoslahti and Pierschbacher, 1987;Hemler, 1990). The integrins interact with specific substrateadsorbed adhesion proteins, often by an Arg-Gly-Asp (RGD) sequence in the adhesion molecules .Increasing attention is being paid to the molecular basis of anti-adhesion . Regulatory substances such as transforming growth factor-ß and interleukin 8 influence adhesion by changing the expression of cell adhesion receptors by the adhering cell (Gimbrone et al., 1989;Heino and Massagu6, 1989) . Matrix-associated molecules such as tenascin (Chiquet-Ehrismann et al., 1988;Lotz et al., 1989;Spring et al., 1989), thrombospondin (Lahav, 1988;Hö6k, 1989), and SPARC (Sage et al ., 1989) inhibit adhesion when adsorbed to a potentially adhesive substrate . We were interested in whether anti-adhesive molecules other than thrombospondin are generated as a result of blood coagulation. Herein we report the purification and characterization ofapotent anti-adhesive globulin fromdextran sulfatetreated human plasma . This globulin inhibits adhesion of a variety of cells to vitronectin-or fibrinogen-coated substrates . Amino acid sequence analysis revealed that the glob-0 The Rockefeller University Press,

Huiman inter-a-trypsin inhibitor (IaTI) is a plasma glycoprotein of Mr 180,000, which has been described as a single polypeptide chain. Recently, however, we proposed that IMTI might be composed of a heavy (H) chain (Mr = 95,000) and a light (L) chain (Mr = 40,000) synthesized by two separate mRNAs. In the present study we have characterized cDNAs for the H chain of Io6TI. These cDNAs collectively covered two sequences (977 and 1450 base pairs in length) with single open reading frames. The deduced amino acid sequences were highly homologous to each other and well matched with partial amino acid sequences obtained from purified serum IaTI. RNA blot analyses of liver RNAs with H-or L-chain cDNAs as probes clearly identified two distinct mRNAs of 3.3 and 1.3 kilobases, which corresponded to H or L chain, respectively. Poly(A)+ RNAs hybrid-selected with H-chain cDNAs coded for polypeptide chains of Mr 90,000-95,000.These results unambiguously establish that IaTI is made of multipolypeptides, possibly including one H and two L chains. The H chain contains potential calcium-binding sites and also regions homologous to the proposed reactive site for thiolproteinase inhibitors. These data indicate that IaTI is a complex, multifunctional protein. mRNAs for both the H and L chains were found only in liver.

, an octapeptide including the HGK motif derived from D5 H , and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro. However, an octapeptide designated P-5m (K487R), in which Lys 487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect. Recombinant GST-D5 H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5 H (K487R) in which Lys 487 was changed to Arg. Furthermore, P-5m (Gly 484 -Lys 491 ) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect. These data strongly indicate that both the HGK motif and lysine residue (Lys 487 ) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo. We tried to identify the HGK motif binding protein on the surface of cancer cells. A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5 H by P-5 (His 479 -Lys 493 ) peptide but not by P-1 (Gly 402 -Lys 420 ) peptide originating from the N-terminal region of D5 H .

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High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of beta-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200 000, ca. 95%) and a minor band of monomeric form (Mr 105 000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110 000), and the monomeric form observed under nonreduced conditions (Mr 105 000) was converted to a heavy chain (Mr 60 000) and a light chain (Mr 50 000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and their derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M beta-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)