Hisae Hori

Synthetic substrates for vertebrate collagenase.

Abstract Seven kinds of 2,4-dinitrophenyl derivatives of hexa- to octapeptides were prepared in order to find a synthetic substrate for vertebrate collagenase. Analysis of the degree of hydrolysis of the peptides by tadpole collagenase and human serum peptidase(s) demonstrated that 2,4 - dinitrophenyl- l -prolyl- l -glutaminyl-glycyl- l -isoleucyl- l -alanyl-glycyl- l -glutaminyl - d - arginine was the best synthetic substrate for collagenase among the peptides tested. The peptide was successfully applied to the assay of collagenase in synovial fluids from patients with rheumatoid arthritis.

Characterization of Dystroglycan‐Laminin Interaction in Peripheral Nerve

Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α‐ and β‐dystroglycan, by posttranslational processing. The 120‐kDa peripheral nerve isoform of α‐dystroglycan binds laminin‐2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan‐laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan‐laminin interaction in peripheral nerve. We demonstrate that (1) α‐dystroglycan is an extracellular peripheral membrane glycoprotein that links β‐dystroglycan in the Schwann cell outer membrane with laminin‐2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α‐dystroglycan in the interaction with laminin.

Differential distribution and expressions of collagens in the cerebral aneurysmal wall

Abstract To investigate the role of collagens in the formation and rupture of cerebral aneurysms, we examined the distribution and synthesis of vascular collagens in the wall of normal human cerebral main trunks and of cerebral aneurysms using immunohistochemistry and in situ hybridization techniques. Fifteen cerebral aneurysmal walls were resected at operation; control cerebral main trunks were obtained from seven autopsy cases. Semiserial sections from the specimens were subjected to immunofluorescence and immunohistochemical staining with antibodies to collagen types I, III, IV, V, VI, desmin and α-smooth muscle actin. In addition, type III collagen mRNA was examined by in situ hybridization. Immunohistochemical study showed that all collagen types were grossly preserved in the aneurysmal wall, although the distribution patterns were different for each collagen. The distribution of major fibrillar collagen types I and III was more diffuse and homogeneous in the luminal layer of the aneurysmal wall than the media of the control artery, although the intensity of immunohistochemical staining was weaker in the abluminal layer of the aneurysmal wall than the adventitia of the control artery. Collagen types IV and V were distributed more sparsely in the luminal layer of the aneurysmal wall than the media of the control artery. Collagen type VI was noted in the luminal as well as the abluminal layer of the aneurysmal wall, whereas it was located exclusively in the adventitia of the control artery. In situ hybridization showed that the signal for collagen type III mRNA on fibroblastic and smooth muscle cells was higher in the aneurysmal walls than the control arteries, suggesting up-regulation of type III collagen transcription in the cerebral aneurysmal wall. The study of the distribution and synthetic regulation of various types of collagen in the aneurysmal wall may be essential for understanding the formation of the aneurysmal wall and its protection against enlargement or rupture.

Entrapment of collagen in a polyacrylamide matrix and its application in the purification of animal collagenases.

Abstract Calf skin collagen with a poor telopeptide region was insolubilized by lattice-entrapment using polyacrylamide gel. The entrapped collagen gel was stable at the pH range of 2.5–9 and unaffected by washing, heating up to 55 °C or wet storage for more than a year. Application of the gel granules in affinity chromatography with tadpole and human synovial collagenases was successfully demonstrated, resulting in an approx. 300-fold purification with a yield of 69% of the initial activity in a single step in the case of tadpole collagenase. The molecular weight of tadpole collagenase thus purified was estimated as approx. 40 000 by molecular-sieve chromatography. The purity of tadpole collagenase prepared by the affinity chromatography was discussed. The gel can be used repeatedly after washing with salt solution in acidic and neutral buffer alternatively.

Immunolocalizations of human gelatinase (type IV collagenase, MMP-9) and TIMP (tissue inhibitor of metalloproteinases) in normal epidermis and some epidermal tumors

The matrix metalloproteinases (MMPs) MMP-2 and MMP-9 (gelatinases) have been suggested as serving an important role in cleaving the basement membrane structure. Tissue inhibitors of metalloproteinases TIMPs (particularly TIMP-1) are known to inhibit MMPs. Based on this background, we raised monoclonal antibodies against human gelatinase (MMP-9) and human recombinant TIMP (TIMP-1), and immunostained these two components in skin from patients with squamous cell carcinoma (SCC), Bowens disease (BD) and keratoacanthoma (KA). MMP-9 showed positive staining mainly in the granular layer of normal epidermis. In some cases of SCC and BD, MMP-9 showed positive staining in the dysplastic lesions even in the basal layer. TIMP showed a thorough positivity in normal epidermis. Unstained regions with this antibody were observed in SCC and BD. These results suggest that an altered staining pattern for MMP-9 and TIMP may be closely related to the malignant transformation of SCC and BD.

Purification of tadpole collagenase and characterization using collagen and synthetic substrates

Tadpole collagenase hydrolyzed native and denatured collagen and synthetic peptides with sequences of 2,4-dinitrophenyl-L-prolyl-L-leucylglycyl-L-isoleucyl-L-alanylglycyl-L-arginie amide and 2,4-dinitrophenyl-L-prolyl-L-glutaminyl-glycyl-L-isoleucyl-L-alanylglycyl-L-glutaminyl-D-arginine. The specific enzyme activity against the latter substrate and collagen fibrils is found to be 933 nmol/min per mg protein and 8440 units (microgram collagen degraded/min), respectively. Optimum pH for the enzyme is 7.5-8.5. A collagenase complex with alpha2-macroglobulin did not hydrolyze collagen fibrils, but digested the synthetic substrates at the Gly-Ile bond. The amino acid composition of the enzyme was determined. Immunoelectrophoresis of the enzyme at pH 8.6 against anti-tadpole collagenase rabbit immunoglobulin G shows a single precipitin line at a position migrating faster than human serum albumin and corresponding to enzyme activity against collagen fibril and synthetic substrates.

IMMUNOHISTOCHEMICAL LOCALIZATION OF TYPE I, II, III, AND IV COLLAGENS IN THE LUNG

Type specific rabbit antibodies to bovine type I, 11, 111, and IV (basement membrane) collagens showing no cross‐reaction with other types of collagen were prepared by cross‐adsorption and diethylamiuoethyl‐cellulose romatography. The antibodies to bovine type I and I11 collagens showed a high cross‐reaction with the corresponding human collagens, but those to type I1 and IV collagens did moderate and no cross‐reactions with human type I1 and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. Both type I and I11 collagens were found to distribute widely in the interstitium of bronchial tree, bronchial

The complete cDNA coding sequence for the bovine Proα2(I) chain of type I procollagen

The complete sequence of the cDNA for the pro alpha2(I) chain of bovine type I procollagen is presented. The encoded amino acid sequence shows 92.0% identity to the human pro alpha2(I) collagen chain.

Production of collagenase inhibitor by the growth cartilage of embryonic chick bone: isolation and partial characterization

Production of collagenase and collagenase inhibitors by the explants of epiphyseal, metaphyseal and diaphyseal regions of embryonic chick limbs during development has been investigated. Collagenase-inhibitory activity was first detected in the culture medium of the diaphyseal region of limbs at stage 36 where cartilage matrix erosion began to occur. However, neither active nor latent collagenase was detected in the media of any regions examined. At stage 38 and later, the maximum production of the inhibitory activity was observed in the explants of metaphyseal region (growth cartilage), while collagenase production was only in the diaphyseal region. Two collagenase inhibitors were isolated and purified approximately 150 fold from the culture medium of stage 38 and 43 metaphyseal regions of limbs by anion- and cation-exchange chromatography followed by gel filtration. The inhibitors are cationic proteins with the same molecular weight of approximately 25,000, but slight difference in molecular charge. They are heat-stable and inhibit collagenases from tadpole skin, chick bone and skin and human granulocytes and gelatinases from human granulocytes and chick skin as well as trypsin, but not Clostridium histolyticum collagenase. A possible function of the inhibitors in multistep regulations of the collagen-degrading enzyme system at the region of osteo-chondral transition is discussed.

Immunolocalization of Human Gelatinase (Type IV Collagenase, MMP-9) and Tissue Inhibitor of Metalloproteinase 1 in Hailey-Hailey and Darier’s Diseases

BACKGROUND The formation of lacunae and acantholysis as well as dyskeratosis are characteristic features of Hailey-Hailey disease (HHD) and Dariers disease (DD). Matrix metalloproteinases (MMPs) and their inhibitors like tissue inhibitors of metalloproteinases (TIMPs) have been thought to play major roles in the tissue metabolism. OBJECTIVE The aim of this study was to investigate the role of MMP-9 and TIMP-1 in HHD and DD. METHODS We examined localizations of these two molecules by immunostaining using specific monoclonal antibodies. RESULTS MMP-9 was positively stained in dyskeratotic or detaching cells around lacunae in HHD and DD. TIMP-1 showed a positive staining pattern throughout the epidermis. CONCLUSION MMP-9 might be involved in the pathophysiological process of HHD and DD in the presence of TIMP-1.