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PubMed1987

Ultrastructural localization of proteoglycans in tissue using cuprolinic blue according to the critical electrolyte concentration method: comparison with biochemical data from the literature.

Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan-protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.

Animals↗

PubMed1992

Cloning and chromosomal location of human alpha 1(XVI) collagen.

We have characterized cDNA clones that encode a newly discovered collagenous polypeptide. A 4-kilobase (kb) cDNA clone was initially isolated by screening a human fibroblast cDNA library with a probe encoding the collagenous domain of the human alpha 3(VI) collagen. Subsequent screening of another fibroblast cDNA library yielded overlapping clones having a total length of 5.4 kb, which contained an open reading frame of 1603 amino acids including a 21-amino acid signal peptide. The predicted polypeptide consists of 10 collagenous domains 15-422 amino acids long, which were interspersed with 11 noncollagenous (NC) domains. Except for a large N-terminal NC11 domain of 312 residues, most of the NC domains were short (11-39 residues) and cysteine-rich. The overall structural arrangement differed significantly from other known collagen chains. Further analysis indicated that the deduced polypeptide exhibited several structural features characteristically seen in members of the fibril-associated collagen, types IX, XII, and XIV. In addition, the cysteine-rich motifs in the NC domains resembled those found in the cuticle collagen of Caenorhabditis elegans. Northern blot analyses showed hybridization of the cDNA to a 5.5-kb mRNA in human fibroblasts and keratinocytes. The gene was localized by in situ hybridization to band p34-35 of human chromosome 1. The data clearly support the conclusion that the cDNA encodes a collagen chain that has not been previously described. We suggest that the cDNA clones encode the alpha 1 chain of type XVI collagen.

Amino Acid Sequence↗

PubMed1993

Molecular cloning of the human alpha 2(IX) collagen cDNA and assignment of the human COL9A2 gene to chromosome 1.

Type IX collagen, a heterotrimer of alpha 1(IX), alpha 2(IX) and alpha 3(IX) chains, is a cartilage-specific fibril-associated collagen. In the process of characterizing genomic clones for the mouse alpha 2(IX) collagen gene four pairs of oligonucleotide primers designed for amplification of murine exon sequences were also utilized to construct cDNA clones for human alpha 2(IX) collagen spanning > 90% of the coding region. The amino acid and nucleotide sequence identities between human and chick are 78% and 71%, respectively. Localization of the COL9A2 gene to human chromosome 1 was subsequently performed using a panel of DNAs from human/rodent somatic cell hybrids.

Amino Acid Sequence↗

PubMed1994

Cellular invasion and collagen type IX in the primary corneal stroma in vitro.

During different stages in the development of the avian cornea, various collagen types have been shown to participate in matrix formation and have been implicated in morphogenesis. One of these is the fibril-associated collagen type IX. This molecule is present when the primary corneal stroma is in a compact state, but rapidly disappears just prior to stromal swelling and its invasion by mesenchymal cells. The temporospatial pattern of the disappearance of type IX collagen in the developing cornea suggests that this molecule may be involved in stabilizing the primary corneal stromal matrix by interacting either with other type IX collagen molecules or with other matrix components. To explore further whether the removal of type IX collagen is involved in stromal swelling, we have employed an in vitro culture system in which swelling of the primary stroma and mesenchymal cell invasion can be experimentally manipulated by culturing chick corneal explants on a Nuclepore filter support in the presence or absence of an associated lens. We have also examined the effect of exogenously added human recombinant tissue inhibitor of metalloproteinases (TIMP-1) on the presence of type IX collagen and cellular invasion. When stage 25-26+ corneal explants were cultured with an associated lens, the primary stroma did not swell; immunohistochemically detectable type IX collagen was still present, and mesenchymal cell invasion failed to occur. Conversely, when the same stages of corneal explants were cultured without an associated lens, the primary stroma swelled; type IX collagen disappeared, and mesenchymal cell migration occurred. Under both conditions, however, the type II collagen of the stroma, which is known to be a component of the striated fibrils, remained clearly detectable and with time even seemed to increase in amount. This result is consistent with the proposition that type IX collagen is one factor involved in maintaining the primary stroma as a compact matrix, possibly by functioning as a bridging/stabilizing factor. When TIMP was added to cultures of corneal explants, type IX collagen remained detectable in focal regions, suggesting that one or more metalloproteinases are involved in the removal of the type IX collagen. In addition, some of these type IX-containing regions contained mesenchymal cells, suggesting that in addition to type IX collagen other factors are likely to be involved in regulating mesenchymal cell migration.

Animals↗

PubMed1991

Collagen family of proteins.

Collagen molecules are structural macro-molecules of the extracellular matrix that include in their structure one or several domains that have a characteristic triple helical conformation. They have been classified by types that define distinct sets of polypeptide chains that can form homo- and heterotrimeric assemblies. All the collagen molecules participate in supramolecular aggregates that are stabilized in part by interactions between triple helical domains. Fourteen collagen types have been defined so far. They form a wide range of structures. Most notable are 1) fibrils that are found in most connective tissues and are made by alloys of fibrillar collagens (types I, II, III, V, and XI) and 2) sheets constituting basement membranes (type IV collagen), Descemet's membrane (type VIII collagen), worm cuticle, and organic exoskeleton of sponges. Other collagens, present in smaller quantities in tissues, play the role of connecting elements between these major structures and other tissue components. The fibril-associated collagens with interrupted triple helices (FACITs) (types IX, XII, and XIV) appear to connect fibrils to other matrix elements. Type VII collagen assemble into anchoring fibrils that bind epithelial basement membranes and entrap collagen fibrils from the underlying stroma to glue the two structures together. Type VI collagen forms thin-beaded filaments that may interact with fibrils and cells.

Amino Acid Sequence↗

PubMed1992

Tissue-specific expression of type XIV collagen--a member of the FACIT class of collagens.

The collagens represent a highly diverse superfamily of extracellular matrix proteins that can be divided into several distinct families. One of the families, called FACIT (fibril-associated collagens with interrupted triple-helices) family, contains molecules that appear to be associated with cross-striated fibrils composed of members of the fibrillar collagen family. We have determined a portion of the primary structure of a recently discovered member of the FACIT family, chicken alpha 1(XIV) collagen, based on cloning and sequencing cDNAs. A synthetic oligopeptide from within the carboxy-terminal non-triple-helical domain of the alpha 1(XIV) chain has been used for generating specific polyclonal antibodies. The antiserum, PS1, recognizes a 220 kDa polypeptide in immunoblots of extracts of chicken skin, tendons, and cartilage. Sequencing of a tryptic peptide generated from purified, immunoreactive material, gives a sequence identical to that derived from cDNA sequencing, providing strong support for the type XIV-specificity of PS1. We have examined the expression of type XIV collagen in developing chick embryos by immunostaining of sections from 12-day-old embryos with PS1 and by Northern blot analysis of RNA from several tissues from both 12- and 17-day-old embryos. The results show that type XIV collagen is prevalent within relatively dense connective tissues such as dermis, tendons, perichondrium, perimysium, the stroma of lungs and liver, and blood vessels.

Amino Acid Sequence↗

PubMed1996

Composition and organization of the extracellular matrix of vein walls: collagen networks.

As there are few recent reports concerning the structure and exact composition of the extracellular matrix from human normal and varicose veins, we carried out comparative immunohistochemical analysis of vessel wall using conventional and confocal laser scanning immunofluorescence techniques. The present report is a rapid review of the structure and function of the 19 known collagen types and our first results on the distribution of collagen types VI, XII and XIV and laminin (glycoprotein from basement membrane) in vein walls. Type VI collagen is concentrated in the sub-endothelium and widely distributed in the media and adventice. For the first time, we demonstrated that both FACIT (fibril-associated) collagens XII and XIV were present in the vein wall, but at different anatomic sites.

Collagen↗

PubMed1991

FACIT collagens: diverse molecular bridges in extracellular matrices.

The collagens form a large family of proteins. Collagen fibrils, composed of staggered arrays of fibrillar collagen molecules (types I, II, III, V and XI), provide a supporting scaffold for extracellular matrices of connective tissues. The non-fibrillar collagens are less abundant than the fibrillar collagens, but it is becoming clear that they have important functions in the matrix. Recently, a group with unique structural characteristics has been defined and named the FACIT (Fibril-Associated Collagens with Interrupted Triple-helices) group. There is evidence that these collagens may serve as molecular bridges that are important for the organization and stability of extracellular matrices.

Animals↗

PubMed1988

Identification of small collagenous proteins with properties of procollagen alpha 1 (I) pN-propeptide in fetal porcine calvarial bone.

Several small collagenous apatite binding (SCAB) proteins have been extracted from the mineralized matrix of fetal porcine calvarial bone. One protein (SCAB 3), released on demineralization of bone with 0.5 M EDTA, appears to represent the alpha 1 pN-propeptide that is normally released during proteolytic processing of type I procollagen. The 28 Kd protein, which stains blue with "Stains-all", is reduced to a 19 Kd fragment by bacterial collagenase digestion, but is not susceptible to cyanogen bromide. The amino acid composition, blocked amino-terminus and immunological properties are all consistent with properties of alpha 1 (I) pN-propeptide. Fractionation on hydroxylapatite in the presence of urea has revealed a nonbinding (SCAB 3a) and a binding (SCAB 3b) form. Extraction of the demineralized matrix of bone with 4 M GuHCl revealed a third form (G2-28) which was similar to SCAB 3a on hydroxylapatite chromatography but showed differences on FPLC "Mono Q" resin. The occurrence of these different forms of pN-propeptide in bone may be of significance in collagen fibril-associated hydroxylapatite formation and in the regulation of osteoblastic function during bone resorption.

Amino Acids↗

PubMed1994

[Proteoglycan in Bruch's membrane of senescence accelerated mouse: localization and age-related changes].

We demonstrated the distribution of sulfated proteoglycans in Bruch's membrane of Senescence Accelerated Mouse histochemically and ultrastructurally using cuprolinic blue in conjunction with specific enzyme treatments and nitrous acid digestion. Two kinds of proteoglycan filaments were observed in the inner and outer collagenous layers, i.e., small collagen fibril-associated filaments (11 nm in average length), and large filaments (32 nm in average length). Intermediate size filaments (25 nm in average length) were seen in the basement membranes of the retinal pigment epithelium and choriocapillaris. Chondroitinase AC treatment eliminated the staining of filaments in the collagenous layers (chondroitin sulfate). Chondroitinase ABC treatment also eliminated the staining of filaments in the collagenous layers (chondroitin sulfate and dermatan sulfate). Nitrous acid eliminated the staining of filaments in both basement membranes (heparan sulfate). Proteoglycans containing chondroitin sulfate and dermatan sulfate were associated uniquely with collagen fibrils. Heparan sulfate proteoglycans were associated with the basement membranes of the pigment epithelium and choriocapillaris. With aging, the thickness of the basement membrane of the choriocapillaris and the staining of the filaments in the basement membranes of the pigment epithelium and choriocapillaris (heparan sulfate proteoglycans) increased. Collagen fibers became disarranged and the staining of both filaments in the collagenous layers decreased. The results of the staining characteristics probably reflect the aging of Bruch's membrane.

Aging↗

PubMed1994

Fibroblast expression of collagens and proteoglycans is altered in aspartylglucosaminuria, a lysosomal storage disease.

Aspartylglucosaminuria (AGU), a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (E.C. 3.5.1.26), is characterised by progressive mental retardation and variable connective tissue signs. The ultrastructure of collagen fibrils in skin of AGU patients is abnormal and their fibroblasts synthesise reduced amounts of collagens [Näntö-Salonen et al. (1984) Lab invest. 51: 464-468]. In this work we measured the steady-state messenger RNA levels of several extracellular matrix components in skin fibroblast cultures of two patients homozygous for the most prevalent mutation (AGUFin) causing the disease in Finland. In confluent cultures the steady-state mRNA concentrations of type I and III collagens were reduced to 0.5-20% of control values. Almost as marked reduction was observed in the mRNA level of biglycan, a small interstitial proteoglycan whereas that of decorin, a closely related, collagen fibril-associated proteoglycan, was increased several-fold. Elevated decorin and decreased biglycan mRNA levels reflected the amounts of the produced corresponding proteoglycans. The differences in the mRNA levels become more pronounced with the time the cells were in culture. Fibronectin mRNA concentrations were similar in AGU and control fibroblasts. Changes in the expression and synthesis of extracellular matrix components might be related to the connective tissue symptoms of the patients.

Acetylglucosamine↗

PubMed1996

Flexilin: a new extracellular matrix glycoprotein localized on collagen fibrils.

We have immunopurified and characterized a new glycoprotein of the extracellular matrix, using a monoclonal antibody obtained after immunization with fibril-associated collagens extracted from bovine tendon. In polyacrylamide gels, the protein migrates at about 350 kDa molecular mass. The protein is insensitive to bacterial collagenase, and no disulfide-linked aggregates could be detected; sugars were stained with periodic acid-Schiff's reagent. Amino acid analysis and sequencing of tryptic peptides failed to detect any similarity with known proteins. By rotary shadowing experiments, the protein was observed as flexible, unbranched structures, approximately 150 nm long, with a small globule at one end. Investigation of the tissue distribution of the protein in fetal bovine tissues by immunofluorescence resulted in labeling in extracellular matrices with loosely packed collagen fibrils, such as the peritendineum, embryonic skin and kidney glomeruli; cornea, cartilage matrix and bone were not labeled. Ultrastructural immunolocalization in dermis and in mesangium of glomeruli showed that the protein always occurred in the vicinity of collagen fibrils. In view of its tissue distribution and molecular shape, we postulate that this protein is important in the properties of the extrafibrillar environment. By reference to its shape as observed by rotary shadowing, we propose the name 'flexilin' for this extracellular matrix glycoprotein.

Amino Acid Sequence↗

PubMed1991

cDNA analysis predicts a cornea-specific collagen.

In the development of chicken corneal stroma, two or more collagens often interact, either as constituents of a single heterotypic fibril or as components of the fibril surface. The latter, fibril-associated collagens, may facilitate interactions between fibrils and the surrounding extracellular matrix or between fibrils themselves. In an effort to isolate putative nonfibrillar collagens that may have such a function, we screened a 13-day embryonic cornea cDNA library under reduced stringency conditions, using a cDNA probe for a collagenous domain of type XII collagen. We isolated a 4.2-kilobase (kb) cDNA that predicts a "collagenous" protein that has three unusual, if not unique, features. (i) The putative polypeptide encoded by this cDNA has a structural arrangement in which numerous stretches of Gly-Xaa-Yaa triplets, typical of collagens, are interrupted by non-Gly-Xaa-Yaa regions. One of the potential triple-helical domains is 246 amino acids long, but most are much smaller, consisting of 15-36 amino acids. Many are very rich in the helix-stabilizing imino acid proline. (ii) Northern blot analyses demonstrated strong cDNA hybridization to a 6.8-kb mRNA whose expression is restricted to the cornea. No hybridization was observed to mRNAs from the nine other tissues used in these analyses, even with extended exposure of the film. (iii) The cDNA contains a short (less than or equal to 425-base-pair) sequence in the 3' untranslated region of the 6.8-kb mRNA that hybridizes to a 7.8-kb mRNA that has a wide tissue distribution.

Amino Acid Sequence↗

PubMed1995

Characterization and partial amino acid sequencing of a 107-kDa procollagen I N-proteinase purified by affinity chromatography on immobilized type XIV collagen.

Procollagen I N-proteinase (EC 3.4.24.14), the enzyme that specifically processes type I and type II procollagens to collagen, was isolated from extracts of fetal calf skin. After two chromatographic steps on concanavalin A-Sepharose and heparin-Sepharose, the semi-purified preparation was used to produce monoclonal antibodies. One reacting antibody was found to recognize not the enzyme itself but type XIV collagen on which the enzyme was bound. This binding, highly sensitive to ionic conditions (plH, salt concentrations) but not affected by non-ionic detergents, was used for affinity chromatography that strongly improved the purification procedure. The enzyme is extensively characterized: 1) it has a molecular mass of 107 kDa as determined by polyacrylamide gel electrophoresis in presence of SDS and of about 130 kDa when estimated by gel filtration on a Sephacryl-S300; 2) in standard assay (pH 7.5, 0.2 M NaCl, 35 degrees C), the activation energy for reaction with amino procollagen type I was 17,000 calories per mole. In the same conditions, Km and Vmax values were, respectively, 435 and 39 nM per hour but varied strongly with pH and salt concentration; 3) the enzyme cleaved the NH2-terminal propeptide of type I procollagen at the specific site, the Pro-Gln bond in the alpha 1 type I procollagen chain; 4) the enzyme contained a high proportion of Gly, Asx, and Glx residues but no Hyp or Hyl; 5) partial amino acid sequences obtained from internal peptides of the enzyme displayed no significant homology with known sequences. The association of procollagen I N-proteinase with a FACIT (fibril-associated collagens with interrupted triple helices) collagen as found here might be of physiological significance.

Amino Acid Sequence↗

PubMed1992

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.

Amino Acid Sequence↗

PubMed1990

Long-range movement and fibril association of type X collagen within embryonic cartilage matrix.

A recent immunoelectron microscopic study of type X collagen in developing cartilage gave results that could be explained by movement of the molecule from one region of the cartilage matrix to another, there becoming associated with preexisting collagen fibrils. In the present study, to test the feasibility of this model we incubated pieces of nonhypertrophic, embryonic chicken sternal cartilage (which has no endogenous type X collagen) in medium with type X collagen and then used immunofluorescence and immunoelectron microscopy to evaluate movement of the molecule through the matrix. The results show that type X collagen molecules can indeed pass through embryonic sternal cartilage matrix and subsequently become fibril-associated.

Animals↗

PubMed1996

Temporal expression of types XII and XIV collagen mRNA and protein during avian corneal development.

Using immunohistochemistry and competitive PCR for collagen types XII and XIV, we have followed the expression of these fibril-associated molecules during development of the avian cornea. By immunofluorescence histochemistry, both molecules are found in the acellular primary stroma and are therefore presumably of epithelial origin. During formation and development of the secondary corneal stroma, which is populated by mesenchymal cells, the molecules generally appear to be spatially segregated from each other. Type XIV collagen is found throughout most of the stroma, and therefore is predominantly a product of stromal fibroblasts. During subsequent compaction of the cornea, an event necessary for corneal transparency, the collagen XIV mRNA level increases dramatically, suggesting that this molecule may play a role in this event. Type XII collagen is more localized, occurring mainly in regions of the secondary stroma where matrices interface, such as where Bowman's membrane and Descemet's membrane abut the orthogonally layered collagen fibrils of the stromal matrix. These interfacial regions are highly stable areas of the cornea as determined previously by protease digestion and thermal denaturation studies. Type XII collagen may be involved in this stabilization.

Animals↗

PubMed1995

Collagen fibrillogenesis in situ: fibril segments undergo post-depositional modifications resulting in linear and lateral growth during matrix development.

Elucidating how collagen fibril growth is regulated is important in determining how tissues are assembled. Fibrils are deposited as segments. The growth of these segments is an important determinant of tissue architecture, stability, and mechanical attributes. Fibril segments were isolated from developing tendons and their structure characterized. The post-depositional changes leading to linear and lateral growth of fibrils also were examined. Segments extracted from 14-day chicken embryo tendons had a mean length of 29 microns. The segments were asymmetric, having a short and a long tapered end. Most of the segments were centrosymmetric with respect to molecular packing. Segments extracted from 12- to 16-day tendons had the same structure, but mean segment length increased incrementally due to the addition of an increasingly large population of longer segments. At 17 days of development there was a precipitous increase in segment length. The morphological data indicate that the increase in length was the result of lateral associations among adjacent segments. Analysis demonstrated that this fibril growth was associated with a significant decrease in fibril associated decorin. Using immunoelectron microscopy, decorin was seen to decrease significantly at 18 days of development. When decorin content was biochemically determined, a decrease also was observed. Decorin mRNA also decreased relative to fibrillar collagen mRNA during the same period. These data support the hypothesis that a decrease in fibril-associated decorin is necessary for fibril growth associated with tissue maturation. Growth through post-depositional fusion allows for appositional and intercalary growth and would be essential for normal development, growth, and repair.

Animals↗