Matrix (Stuttgart, Germany)最新文献

The carbohydrate content of mouse nidogen predicts the occupation of two N- and about seven O-linked acceptor sites. The corresponding oligosaccharides were examined by sequential exoglycosidase digestions. The data indicate N-linked substitutions by several bi-, tri- and tetraantennary complex types of oligosaccharides which are further modified by additional lactosamines and terminal α-galactose and/or sialic acid. Mannose-rich oligosaccharides were of low abundance. O-linked structures included a di- and tetrasaccharide core structure that were in addition sialylated and may be similar to structures found in fetuin. Evidence is provided that the two sequence-predicted asparagine acceptors are almost fully substituted. Sequence analysis of tryptic peptides identified Thr-271, Ser-303, Thr-309, Thr-317, Thr-320, Thr-892 and Thr-905 as the most likely sites for galactosamine substitutions. These residues are located in the flexible link connecting the N-terminal globular domains G1 and G2 of nidogen and at the border between the rod and the C-terminal globe G3. Four of them showed Pro in the −1 or + 3 position. All these Ser, Thr and Pro residues but not the N-linked attachment sites are identical in human nidogen.

In this study we describe a human sulfated 30 kD protein (sp30) that is recognized by a monoclonal antibody raised against human vitronectin (mAb 8E6). Another monoclonal antibody raised against human vitronectin, mAb MaSp, and a polyclonal antiserum against vitronectin did not react detectably with sp30. Sp30, unlike vitronectin, is synthesized by a variety of non-hepatic human cell lines in culture, including cells of lymphoid origin. It is synthesized in sulfated form as indicated by metabolic labeling of MG-63 human osteosarcoma cells with ³⁵SO₄. Sp30 is an extracellular matrix protein as indicated by its association with the matrix of MG-63 cells after removal of the cells with EDTA and its fibrillar pattern by immunofluorescence of non-permeabilized confluent MG-63 cell monolayers detected with mAb 8E6. This antibody also stained short fibrils in human embryonic tissue. This pattern was distinct from the fainter diffuse staining obtained with mAb MaSp and the polyclonal antiserum to vitronectin, suggesting that the 8E6 staining in embryonic tissues was mostly due to sp30 rather than vitronectin. A polyclonal antiserum against bovine microfibril associated glycoprotein (MAGP) precipitated a [³⁵SO₄]-30 kD protein from [³⁵SO₄]-labeled MG-63 medium that co-migrated with a band precipitated by mAb 8E6. Double-labeling immunofluorescence studies of embryonic tissues showed an identical distribution of anti-bovine MAGP antiserum and mAb 8E6 staining. These data indicate that sp30 is the human homolog of bovine MAGP. Distinction between sp30 and vitronectin will be important in ascertaining the localization and function of both proteins. The findings that sp30 is sulfated and synthesized and secreted by a variety of cells in culture should aid in defining its role in microfibrillogenesis. That sp30 is secreted by cells of lymphoid origin suggests that it might also have a heretofore unsuspected role in immune responses.

Four hybrid cell lines producing monoclonal antibodies (designated AC2, AH12, DB10 and DD11) were derived from mice immunized with the large chondroitin sulphate proteoglycan isolated and purified from the bovine temporomandibular joint disc. The epitopes were partially characterized by enzyme-linked immunosorbent assays and staining patterns on immunoblots of intact proteoglycans and digests made with glycosidases and proteinases. All four monoclonal antibodies appeared to recognize some form of keratan sulphate although the epitopes for two (AC2 and DD11) were probably identical. One antibody (AH12) showed almost no reactivity with corneal keratan sulphate but stained a small keratan sulphate proteoglycan extracted from the disc, in addition to the large chondroitin sulphate proteoglycan. These antibodies were used for immunohistochemical staining of sections of the disc and showed that keratan sulphate associated with the large chondroitin sulphate proteoglycan was concentrated inside and away from the periphery of the structure but close to the inferior and superior surfaces, in a pattern which may reflect the adaptation of the extracellular matrix to the mechanical stresses placed on it by mastication.

The effects of transforming growth factor-β1 (TGF-β1) and all-trans retinoic acid (RA) on human dermal fibroblast proliferation and collagen production were investigated in ‘attached’ and ‘detached’ dermal equivalent (DEs) systems compared with fibroblasts grown in monolayers. The combined effects of TGF-β1 with epidermal growth factor (EGF) on fibroblast proliferation and collagen production were also studied. Fibroblast proliferation was stimulated 0.7-fold by TGF-β1 in attached DEs and 0.3-fold in detached DEs compared with untreated control DEs. RA stimulated fibroblast proliferation 1.1-fold in attached DEs and 0.6-fold in detached DEs. Neither TGF-β1 nor RA had a significant effect on fibroblast proliferation in monolayer cultures. In the presence of EGF, the action of TGF-β1 on fibroblast proliferation was slightly suppressed in attached DEs. At 5 ng/ml TGF-β1, collagen production was stimulated 6.1-fold in attached DEs, by 3.5-fold in monolayer at 4 ng/ml and 2.9-fold in detached DEs at 1 ng/ml. RA at 5 × 10⁻¹⁰ M to 5 × 10⁻⁶ M stimulated collagen production in all three systems. The stimulation of collagen production by 5 ng/ml TGF-β1 was suppressed by EGF in both attached DEs and monolayer culture. Fibroblasts in attached DEs had elongated, bipolar morphology with a tendency to line up in the same direction. Fibroblasts in detached DEs were randomly distributed and exhibited stellate morphology. These data indicate that the extracellular matrix strongly influences the actions of growth factors and of RA on dermal fibroblasts. When stimulated with TGF-β1 collagen production in attached DEs was higher than that by fibroblasts in monolayer culture and detached DEs. The attached and detached DEs offer a model which resembles the in vivo situation more closely than monolayer culture with respect to collagen production and fibroblast proliferation and morphology.

The entire primary structure of the murine α1(VI) collagen chain was deduced from cloned cDNA. The predicted polypeptide consists of 1025 amino acids and shows extensive homology with the corresponding human and chicken chains. A genomic clone isolated with a cDNA probe was found to contain about 13 kilobases of the 5′-flanking region and the first and second exon, coding for the 5′-untranslated sequence and signal peptide and part of the N-terminal portion of the mature protein, respectively. Polymerase chain reaction and primer extension analyses revealed two major and several minor transcription start sites distributed over 76 base pairs (bp). The region just upstream of the transcription initiation sites lacks canonical TATA and CAAT boxes and Sp1 binding sites, but contains putative binding sites for other transcription factors and a 90-bp polypyrimidine tract with elements of dyad symmetry. Chimeric constructs were derived from different fragments of the 5′-flanking genomic region and the chloramphenicol acetyltransferase (CAT) gene and expression of the reporter gene was assayed following transfection of various cell types. A construct containing sequences extending from −215 to +41 directed high levels of CAT expression. The data indicate that this region harbours a functional promoter.

Acid-soluble collagen from rat skin was modified by active oxygen in vitro, and properties of the modified collagen as a substratum for fibroblasts were studied. When collagen was treated with ascorbate-copper ion systems, cross-linking and a little degradation occurred rapidly. The cells attached but spread poorly on the modified collagen gel as compared with on the untreated collagen gel. On the other hand, when collagen was treated with H₂O₂-copper ion systems, only degradation of collagen molecule occurred rapidly. This treatment did not affect the attachment and spreading of the cells on the collagen gel, but when the incubation was continued for a long time, the cells migrated actively and gathered. Thymidine incorporation by the cells was suppressed on both modified collagen gels as compared with that on untreated collagen gel, and the extent of the suppression on the H₂O₂-copper-treated collagen was larger than that on the ascorbate-copper-treated collagen. These results indicate that the active oxygen-induced cross-linking and degradation significantly alter properties of collagen as a substratum for fibroblasts.

A protein (M_r 24 K) that co-purifies with porcine skin lysyl oxidase (M_r 34 K) has been isolated and characterised. Five variants of the 24 K protein were identified by Mono Q ion-exchange FPLC, as were four variants of lysyl oxidase. Amino acid analysis and partial sequencing revealed near identity of a 36-residue CNBr peptide from porcine skin lysyl oxidase to corresponding regions of the putative lysyl oxidase precursor derived from rat and human cDNA. The 24 K protein was found to be unrelated to lysyl oxidase, but comparison with a protein sequence database showed it to be the same as a recently described protein from bovine skin that is associated with dermatan sulphate proteoglycans. The 24 K protein is relatively rich in tyrosine, and isoelectric focussing shows it to be acidic, with pI's in the range 4.1 to 4.4. In view of these properties, we propose the name TRAMP (Tyrosine Rich Acidic Matrix Protein) to identify this protein. Though TRAMP appears not to be glycosylated, several experiments indicate the presence of sulphotyrosine residues. When assayed using an elastin substrate, the activity of lysyl oxidase is unaffected by TRAMP.

The turnover of proteoglycans was studied in explant cultures of mature bovine articular cartilage. The aim of the study was to compare the in vitro turnover rates of newly synthesized proteoglycans and endogenous proteoglycans. Cartilage was maintained in the presence of various serum concentrations in order to determine the conditions of steady-state proteoglycan metabolism. The steady state was achieved in medium containing 20% fetal calf serum.

The proteoglycan synthesis rate and the half-life of labeled proteoglycans in steady-state cultures were used to calculate the size of the metabolic pool of newly synthesized proteoglycans in steady state. This metabolic pool was shown to be equal to the total amount of proteoglycans in the matrix. It is concluded that all of the proteoglycans in the matrix have the same half-life in vitro.

Taking another approach, aggrecan was isolated from the cartilage and the medium of steadystate cultures prelabeled with [³⁵S] sulfate. The specific activity of the glycosaminoglycans from cartilage aggrecan were compared with that of glycosaminoglycans from medium aggrecan. These proved to be the same throughout the culture period. This shows that newly synthesized aggrecan and endogenous aggrecan have the same turnover rate in vitro.

The significance of explant culture systems for the study of proteoglycan turnover is discussed.

Two members of the matrix metalloproteinase family which can cleave native types I, II and III triple helical collagens are collagenases from fibroblasts and neutrophils. These enzymes are the products of different genes which share structural motifs but are only 57% identical. In this study, we determined the site of cleavage in the α1(I) chains and showed that the neutrophil collagenase acted at the same site as the fibroblast collagenase. We also used collagens as substrates which were generated by site-directed mutagenesis of the murine Col1a1 gene and found that the pattern of susceptibility to cleavage by purified neutrophil collagenase was indistinguishable from that previously described for the fibroblast collagenase. Collagens containing substitutions of Pro for Ile-776 (P₁) were not cleaved; whereas those containing substitutions of Met for Ile-776 were cleaved. Type I collagen which contained α1(I) chains in which there were double substitutions of Pro for Gln-774 (P₂) and Ala-777 (P₂′) were also not cleaved. These type I collagens contained wild type α2(I) chains as well as mutant α1(I) chains in the mixed helical trimers; the α2(I) chain in the trimers containing the resistant α1(I) chains were also not cleaved by the neutrophil collagenase.

Type X collagen, a homotrimer of α1(X) polypeptide chains, is specifically expressed by hypertrophic chondrocytes in regions of cartilage undergoing endochondral ossification. We have previously characterized a genomic clone containing the major part of the mouse type X collagen gene (col10a1) and assigned the locus for co110al to mouse chromosome 10 (Apte et al., Eur. J. Biochem. 224: 217–224, 1992). In this paper, through additional characterization of cDNA and genomic clones, we describe the complete organization of the col10a1 gene. The col10a1 gene is 7.2 kb in size and, as in the chick, col10a1 gene transcripts are generated from only three exons. Exon 1 encodes only the 5′ untranslated (5′ UT) region of the mRNA and is separated from exon 2 by an intron 562 by in length. Exon 2 (169 bp) encodes 15 by of 5′ UT message, the translation start plus 17-amino acid residues of the putative signal peptide, and 33 1/3 codons of the putative N-terminal non-collagenous (NC2) domain of type X collagen. Exon 3 is 2854 by in size and encodes 4 2/3 codons of the NC2 domain, the entire collagenous domain of 463 residues (COL), the entire C-terminal non-collagenous (NC1) domain (161 amino acid residues) and the entire 965 by 3′ untranslated (3′ UT) sequence of the mRNA. Two TATA boxes are present in tandem in the col10a1 promotor. Both TATA boxes are active in transcription, generating two populations of transcripts with different 5′-termini; the longer transcript is of low abundance and is detectable only by PCR in newborn mice. The col10a1 promotor contains a CCAAT box as well as other consensus sequence elements required for binding of potential transcription factors. Characterization of the col10a1 gene provides data essential for studies of the regulation of type X collagen expression during mammalian endochondral bone growth and development. Knowledge of the complete structure of the mouse typeX collagen gene will also be useful for the investigation of type X collagen gene abnormalities in murine chondrodysplasias and for the generation of transgenic mice.