Developmental distribution of collagen type XII in cartilage: association with articular cartilage and the growth plate.

Collagen type XII is a member of the fibril-associated collagens and is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains. Previous studies suggested that collagen XII is a component of cartilage but little is known about its spatial-temporal distribution. This study uses a polyclonal antibody to the purified NC3 domain to investigate its developmental distribution in rat forelimb. Collagen XII was present at the joint interzone on embryonic day 16 (E16d) and restricted to the presumptive articular cartilage by E18d. Labeling of the articular surface intensified as development progressed postnatally (day 1 [1d] to 28d) and extended approximately six cell diameters deep. In juvenile rats, collagen XII antibodies also labeled the longitudinal and transverse septa of stacked chondrocytes in the growth plate. However, collagen XII was not associated at any developmental stage with the cartilaginous secondary ossification center and was only weakly expressed in epiphyseal cartilage. Ultrastructural localization of the NC3 domain epitope showed labeling of the surface of collagen II fibrils both in tissue and in isolated fibrils. The results presented provide further evidence that articular cartilage differs substantially from the underlying epiphyseal cartilage and that different chondrocytic developmental fates are reflected in the composition of their extracellular matrix starting early in development. In addition, collagen XII was distributed in areas of cartilage with more organized fibril orientation and may have a role in promoting alignment or stabilizing such an organization, thereby creating a matrix capable of withstanding load-bearing forces.

Antiproliferative effects of insulin-like growth factor-binding protein-3 in mesenchymal chondrogenic cell line RCJ3.1C5.18. relationship to differentiation stage.

Chondrogenesis results from a complex equilibrium between chondrocyte proliferation and differentiation. Insulin-like growth factors (IGFs) have a crucial role in chondrogenesis, but their mechanisms of action are not well defined. IGF-binding protein-3 (IGFBP-3) is the major carrier for circulating IGFs in postnatal life, and has been shown to have IGF-independent effects on proliferation of several cancer cell lines. In this study, we have evaluated the IGF-independent and -dependent effects of IGFBP-3 on chondrocyte proliferation and the relationship of these effects with chondrocyte differentiation stage. We used the RCJ3.1C5.18 nontransformed mesenchymal chondrogenic cell line, which, over 2 weeks of culture, progresses through the differentiation pathway exhibited by chondrocytes in the growth plate. We demonstrated that IGFBP-3 inhibited, in a dose-dependent manner (1-30 nm), the proliferation of chondroprogenitors and early differentiated chondrocytes, stimulated by des-(1-3)-IGF-I and longR(3)-IGF-I (IGF-I analogs with reduced affinity for IGFBP-3), and by insulin and IGF-I. In terminally differentiated chondrocytes, IGFBP-3 retained the ability to inhibit cell proliferation stimulated by IGF-I, but had no effect on cell growth stimulated by insulin, or des-(1-3)-IGF-I or longR(3)IGF-I. By monolayer affinity cross-linking, we demonstrated a specific IGFBP-3-associated cell-membrane protein of approximately 20 kDa. We determined that IGFBP-3 has an antiproliferative effect on chondrocytes and, that this effect is related to the differentiation process. In chondroprogenitors and early differentiated chondrocytes, antiproliferative effect of IGFBP-3 is mainly IGF-independent, whereas, following terminal differentiation this effect is IGF-dependent.

Col2-GFP reporter mouse--a new tool to study skeletal development.

Transgenic mice were generated that harbor a Col2-GFP reporter that marks chondrocytes and their immediate precursors during skeletal development. Cells engaged in chondrogenesis were identified by conventional fluorescence microscopy and confocal optical sectioning within their native environments in live embryos and in thick tissue slices. The use of these mice offers a novel approach for studying the role of chondrocytes in skeletal development.

LMX1B transactivation and expression in nail-patella syndrome.

Lmx1b, a member of the LIM homeodomain protein family, is essential for the specification of dorsal limb fates at the zeugopodal and autopodal level in vertebrates. We and others have shown that a skeletal dysplasia, nail-patella syndrome (NPS), results from mutations in LMX1B. While it is a unique mesenchymal determinant of dorsal limb patterning during vertebrate development, the mechanism by which LMX1B mutations generate the NPS phenotype has not been addressed at a transcriptional level or correlated with its spatial pattern of gene expression. In this study, in situ hybridizations of Lmx1b on murine limb sections reveal strong expression in dorsal mesenchymal tissues (precursors of muscle, tendons, joints and patella) and, interestingly, also in anterior structures of the limb, explaining the anterior to posterior gradient of joint and nail dysplasia observed in NPS patients. Transfection studies showed that both the LIM domain-interacting protein, LDB1, and the helix-loop-helix protein, E47/shPan1, can regulate LMX1B action. While co--transfections of E47/shPan1 with LMX1B result in a synergistic effect on reporter activity, LDB1 down-regulated LMX1B-mediated transactivation irrespective of E47/shPan1. Mutant LMX1B proteins containing human mutations affecting each of the helices or the N-terminal arm of the homeodomain abolished transactivation, while LIM B and truncation mutations retained residual activity. These mutations fail to act in a dominant-negative manner on wild-type LMX1B in mixing studies, thereby supporting haploinsufficiency as the mechanism underlying NPS pathogenesis.

Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice.

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high-affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9-FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.

Differential effects of fibroblast growth factor (FGF) 9 and FGF2 on proliferation, differentiation and terminal differentiation of chondrocytic cells in vitro.

Fibroblast growth factor (FGF) 9 was compared with FGF2 in its ability to influence proliferation, differentiation, terminal differentiation and apoptosis in a rat calvaria-derived cell line (RCJ 3.1C5.18) that spontaneously undergoes chondrocyte differentiation in vitro. Like FGF2, FGF9 promoted proliferation, but to a lesser extent. In contrast to FGF2, which blocked chondrocytic differentiation, FGF9 had no effect on differentiation but inhibited terminal differentiation. FGF9 also stimulated expression of the mitotic inhibitor p21 to a greater extent than FGF2. Neither ligand influenced apoptosis. The results indicate that FGF9 could account for many of the physiological responses attributed to FGF-receptor activation in the growth plate.

Chondrocyte differentiation in a rat mesenchymal cell line.

We used a combination of morphologic and histochemical methods to demonstrate that rat calvaria-derived mesenchymal cells, RCJ 3.1C5. 18, in culture progress through the differentiation pathway exhibited by chondrocytes in the endochondral growth plate. The cells were grown either as monolayer or suspension cultures. Subconfluent monolayer cultures did not express markers typical of chondrocyte phenotypes. However, after reaching confluency the cells formed nodules of chondrocytic cells separated by cartilage-appearing matrix and encapsulated by fibroblast-like cells. Suspension culture produced cell aggregates with similar characteristics. Matrix in both the nodules and aggregates stained for collagen Types II and XI and aggrecan, and some cells displayed a distinctive pericellular matrix that stained for Type X collagen. Mineralization was evident in older cultures. By electron microscopy, most cells in the aggregates appeared as typical chondrocytes. However, some larger cells were surrounded by a "mat" of matrix comprised of hexagonal arrays of dense nodules interconnected by a filamentous network. Immunogold localization confirmed the presence of collagen Type X in this matrix. Analysis of markers of chondrocyte differentiation and terminal differentiation over time showed that these markers were acquired sequentially over 2 weeks of culture. This model system will be useful to study the regulation of various steps in the chondrocyte differentiation pathway.

Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment.

Stable attachment of external epithelia to the basement membrane and underlying stroma is mediated by transmembrane proteins such as the integrin alpha6beta4 and bullous pemphigoid antigen 2 within the hemidesmosomes along the basolateral surface of the epithelial cell and their ligands that include a specialized subfamily of laminins. The laminin 5 molecule (previously termed kalinin/nicein/epiligrin) is a member of this epithelial-specific subfamily. Laminin 5 chains are not only considerably truncated within domains III-VI, but are also extensively proteolytically processed in vitro and in vivo. As a result, the domains expected to be required for the association of laminins with other basement membrane components are lacking in the mature laminin 5 molecule. Therefore, the tight binding of laminin 5 to the basement membrane may occur by a unique mechanism. To examine laminin 5 in tissue, we chose human amnion as the source, because of its availability and the similarity of the amniotic epithelial basement membrane with that of skin. We isolated the laminin 5 contained within the basement membrane of human amnion. In addition to monomeric laminin 5, we find that much of the laminin 5 isolated is covalently adducted with laminin 6 (alpha3beta1gamma1) and a novel laminin isotype we have termed laminin 7 (alpha3beta2gamma1). We propose that the association between laminin 5 and laminins 6 and 7 is a mechanism used in amnion to allow stable association of laminin 5 with the basement membrane. The beta2 chain is seen at the human amniotic epithelial-stromal interface and at the dermal-epidermal junction of fetal and adult bovine skin by immunofluorescence, but is not present, or only weakly present, in neonatal human skin.

Basement membrane proteins kalinin and nicein are structurally and immunologically identical.

BACKGROUND: The purpose of this investigation was 2-fold: (a) to compare two recently described proteins, the anchoring filament protein, kalinin and the hemidesmosome-associated protein, nicein (formerly called BM-600) which are both absent in junctional epidermolysis bullosa (JEB) Herlitz's disease; (b) to further define the structural defect in JEB Herlitz's disease. EXPERIMENTAL DESIGN: Cultured keratinocytes were analyzed with monoclonal antibodies (mAbs) against kalinin and nicein by indirect immunofluorescence. These mAbs were also used to immunoprecipitate radiolabeled proteins from keratinocyte cultures and to immunoaffinity purify proteins from keratinocyte conditioned culture medium. The precipitated or purified products were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, partial V8 protease digestion, and rotary shadowing. RESULTS: Kalinin and nicein mAbs show identical immunofluorescent staining patterns on cultured keratinocytes. Kalinin and nicein mAbs immunoprecipitate peptides from radiolabeled normal human keratinocyte cell and medium fractions that are electrophoretically identical. Partial V8 protease digestion patterns of the reduced 140 kilodalton peptides precipitated by nicein and kalinin mAbs are identical. Kalinin (like nicein) is absent from JEB Herlitz keratinocyte conditioned medium although K-laminin, another anchoring filament component, is present in these cultures. Kalinin, purified from conditioned keratinocyte medium by antibody affinity chromatography with K140-Sepharose (mAb against kalinin) and nicein purified from conditioned keratinocyte medium with GB3-Sepharose (mAb against nicein) are electrophoretically identical by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunologically identical by immunoblotting using kalinin and nicein mAbs. Rotary shadowed images of kalinin and nicein molecules are identical. CONCLUSIONS: We demonstrate that kalinin and nicein are identical by biochemical and immunologic analysis. We also verify that kalinin, like nicein, is absent in the conditioned medium of cultured JEB Herlitz keratinocytes, although another anchoring filament protein, K-laminin, is secreted by these cultures. These results correlate with previous immunofluorescent findings that show that while kalinin or nicein is absent in basement membranes of individuals with JEB Herlitz's disease, K-laminin appears to be present.

The dermal-epidermal junction of human skin contains a novel laminin variant.

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.

Identification and partial purification of a large, variant form of type XII collagen.

A large, alternate form of type XII collagen has been identified in cultures of the human epidermoid cell line WISH. This form, designated XIIA, is comprised of alpha chains that are approximately 90 kDa larger than the 220-kDa alpha chain previously characterized in extracts of fetal chicken and bovine tissues. Results from both collagenase digestion and rotary shadow analysis of partially purified material show that the increase is due to a larger NC3 domain. While both the large (XIIA) and the small (XIIB) forms of type XII collagen are identified in pulse-chase radiolabeling of fetal bovine skin explant culture, they are not related in a precursor-product fashion. Inhibition studies with alpha, alpha'-dipyridyl indicate that proper folding of the collagen helix is required for complete assembly and secretion of type XIIA in WISH cell culture. The 310-kDa alpha 1A chain is likely to represent the bovine equivalent of a second translation product, estimated to be 340 kDa, predicted from analysis of one complete chick cDNA sequence. Additionally, the amino-terminal amino acid sequence of the 220-kDa bovine alpha 1B chain was determined. This sequence is very near a potential alternate splice site predicted from analysis of chicken type XII cDNA.

Characterization of collagen types XII and XIV from fetal bovine cartilage.

The structurally related type XII-like collagen molecules TL-A and TL-B were recently identified in fetal bovine epiphyseal cartilage and subsequently shown to be collagen types XII and XIV, respectively. By indirect immunofluorescent staining of cartilage using monoclonal antibodies to the NC3 domains of each molecule, it was shown that type XII collagen was present predominantly around cartilage canals, the articular surface, subperichondrial margins, and the perichondrium, was less so in the remaining cartilage matrix, and was absent from the growth plate region. In the permanent cartilage of trachea, type XII stained somewhat more intensely in the margins beneath the loose connective tissue. Type XIV collagen localized more uniformly throughout the articular cartilage and was also absent from the growth plate region, whereas in tracheal cartilage, its distribution was similar to type XII. We have characterized the structure of these cartilage molecules and compared them with those from fetal bovine skin. Extraction of cartilage with 1 M NaCl and differential NaCl precipitation yields a fraction enriched for these two collagens. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with monoclonal antibodies to the large amino-terminal non-triple-helical domain, NC3, revealed the presence in cartilage of two forms of type XII collagen: type XIIB, the molecule previously identified in chick and bovine tissues, and type XIIA, a much larger form equivalent to the molecule recently identified in WISH-transformed epithelial cell culture medium (Lunstrum, G. P., McDonough, A. M., Marinkovich, M. P., Keene, D. R., Morris, N. P., and Burgeson, R. E. (1992) J. Biol. Chem. 267, 20087-20092). Digestion with bacterial collagenase shows that the increased mass is present in the NC3A domain. Additional purification by velocity sedimentation and observation of rotary-shadowed images demonstrates molecules with extended non-triple-helical arms approximately 80 nm in length analogous to the WISH cell molecules. Electrophoretic mobilities of bands corresponding to type XIIA, but not type XIIB, are sensitive to chondroitinase ABC, indicating that type XIIA is a chondroitin sulfate proteoglycan and that modification occurs predominantly within the NC3A domain distal to NC3B. Neither type XIIB from skin nor type XIIA from WISH cells are chondroitinase-sensitive. By similar analysis, a portion of the type XIV collagen chains in cartilage was also sensitive to chondroitinase digestion. Chondroitin sulfate is apparently not located on its NC3 domain. As in skin, collagen types XII and XIV have subtly different distributions within cartilage and type XII may have a tissue-specific structure.

The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor.

Kalinin, a recently characterized novel protein component of anchoring filaments, has been shown to be involved in keratinocyte attachment to culture substrates and to dermis in vivo, and to exist in keratinocyte-conditioned culture medium in two heterotrimeric forms of 440 and 400 kDa (Rousselle, P., Lunstrum, G.P., Keene, D.R., and Burgeson, R.E. (1991) J. Cell Biol. 114, 567-576). This study demonstrates that kalinin is initially synthesized in a cell-associated form estimated to be 460 kDa. By second dimension reduced electrophoresis, V8 protease digestion, and immunoblot analysis, we demonstrate that the cell form contains nonidentical subunits of 200, 155, and 140 kDa. The 440-kDa medium form is derived from the cell form by extracellular processing of the 200-kDa subunit to 165 kDa, a step which also occurs in skin organ culture. The 400-kDa form is derived from the 440-kDa form by extracellular processing of the 155 kDa-subunit to 105 kDa. The cell form is secreted by keratinocytes, deposited onto culture substratum, and is the form which facilitates attachment and adhesion of growing and spreading keratinocytes. It is also the form initially synthesized in skin organ culture. Kalinin purified from tissue, which appears to facilitate epithelial-mesenchymal cohesion in vivo, is closely related to the 400-kDa medium form purified from culture.

Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.

Basal keratinocytes attach to the underlying dermal stroma through an ultrastructurally unique and complex basement membrane zone. Electron-dense plaques along the basal surface plasma membrane, termed hemidesmosomes, appear to attach directly to the lamina densa of the basement membrane through fine strands, called anchoring filaments. The lamina densa is secured to the stroma through a complex of type VII collagen containing anchoring fibrils and anchoring plaques. We have identified what we believe is a novel antigen unique to this tissue region. The mAbs to this antigen localize to the anchoring filaments, just below the basal-dense plate of the hemidesmosomes. In cell culture, the antigen is deposited upon the culture substate by growing and migrating human keratinocytes. Addition of mAb to the cultures causes the cells to round and detach, but does not impair them metabolically. Skin fragments incubated with antibody extensively de-epithelialize. These findings strongly suggest that this antigen is intimately involved in attachment of keratinocytes to the basement membrane. This antigen was isolated from keratinocyte cultures by immunoaffinity chromatography. Two molecules are observed. The most intact species contains three nonidentical chains, 165, 155, and 140 kD linked by interchain disulfide bonds. The second and more abundant species contains the 165- and 140-kD chains, but the 155-kD chain has been proteolytically cleaved to 105 kD. Likewise, two rotary-shadowed images are observed. The larger of the two, presumably corresponding to the most intact form, appears as an asymmetric 107-nm-long rod, with a single globule at one end and two smaller globules at the other. The more abundant species, presumably the proteolytically cleaved form, lacks the distal small globule. We propose the name "kalinin" for this new molecule.

Identification and partial characterization of two type XII-like collagen molecules.

We have identified two distinct collagenous macromolecules in extracts of fetal bovine skin. Each of the molecules appears to contain three identical alpha-chains with short triple-helical domains of approximately 25 kD, and nontriple-helical domains of approximately 190 kD. Consistent with these observations, extracted molecules contain a relatively short triple-helical domain (75 nm) and a large globular domain comprised of three similar arms. Despite these similarities, the purified collagenase-resistant domains are distinguished by a number of criteria. The globular domains can be chromatographically separated on the basis of charge distribution. Peptide profiles generated by V8 protease digestion are dissimilar. These molecules are immunologically unique and have distinct distributions in tissue. Finally, rotary shadow analysis of purified domains identifies size and conformation differences. Structurally, the molecules are very similar to type XII collagen, but differ in tissue distribution, since both these molecules are present in cartilage, while type XII is reported to be absent from that tissue.

Two type XII-like collagens localize to the surface of banded collagen fibrils.

Two recently identified collagen molecules, termed twelve-like A and twelve-like B (TL-A and TL-B) have properties similar to type XII collagen. These molecules have been localized in human and calf tissues by immunoelectron microscopy. The observations strongly suggest that both molecules are located along the surface of banded collagen fibers. The epitopes recognized by the antibodies are contained in large, nontriple-helical domains at one end of the collagen helix. The epitopes are visualized at a distance from the surface of the banded fibers roughly equal to the length of the nonhelical domains, suggesting that the nonhelical domains extend from the fibril, while the triple-helical domains are likely to bind directly to the fibril surface. Occasionally, both TL-A and TL-B demonstrate periodic distribution along the fibril surface. The period corresponds to the primary interband distance of the banded fibrils. Not all fibrils in a fiber bundle are labeled, nor is the labeling continuous along the length of labeled fibrils. Simultaneous labeling of TL-A and type VI collagen only rarely shows colocalization, suggesting that TL-A and TL-B do not mediate interactions between the type VI collagen beaded filaments and banded collagen fibrils. Also, interfibrillar distances are approximately equivalent in the presence and absence of these type XII-like molecules. While the results do not directly indicate a specific function for these molecules, the localization at the fibril surface suggests that they mediate interactions between the fibrils and other matrix macromolecules or with cells.

The relationship of the biophysical and biochemical characteristics of type VII collagen to the function of anchoring fibrils.

Type VII collagen is a major component of anchoring fibrils, which are 800-nm-long centrosymmetrically cross-banded fibrils that are believed to secure the attachment of certain epithelial basement membranes to the underlying stromal matrix. The ultrastructure of the anchoring fibrils is highly variable, suggesting that the fibrils are flexible. Flexibility measurements along the length of the triple-helical domain of type VII procollagen indicate that major flexible sites correlate well with known discontinuities in the (Gly-X-Y)n repeating sequence. Therefore, the helical disruptions may account for the tortuous shapes of anchoring fibrils observed ultrastructurally. The centrosymmetrical banding pattern observed for anchoring fibrils results from the unstaggered lateral packing of antiparallel type VII collagen dimers that form these structures. This antiparallel arrangement is specified by disulfide bonds formed at the margins of a 60-nm overlap of the amino termini. As long as these disulfide bonds remain intact, they protect the amino-terminal overlapping triple helices from collagenase digestion. This disulfide-bonded pair of triple helices is termed C-1. Large nonhelical domains (NC-1) extend from both ends of the anchoring fibrils and are believed to interact with the basement membrane or with anchoring plaques. Rotary shadowing of the NC-1 domains showed trident-like shapes, suggesting that a single alpha-chain contributed the structure of each arm and that the three arms were extended. Biochemical and biophysical analyses of NC-1 domains independently confirm these suggestions and imply that the arms of NC-1 domains are identical and individually capable of interactions with basement membrane components, potentially allowing trivalent interaction of type VII collagen with various macromolecules.

Drosophila basement membrane procollagen IV. I. Protein characterization and distribution.

A collagen was isolated from Drosophila E85, Schneider line 2L and Kc cell cultures. The purified protein was characterized and antibodies were raised against it. Immunofluorescence microscopy locates this material to the regions of basement membranes of Drosophila embryos, larvae, and adults. The molecules are mostly, or entirely, homotrimers of one polypeptide chain linked by interchain disulfide bonds. The partial amino acid sequences of a cyanogen bromide cleavage product of this chain are identical with a part of the virtual translation product of the Drosophila pro alpha 1(IV) nucleotide sequence that is reported in the accompanying paper. This gene is at Drosophila chromosome location 25C and was identified by the high homology of one part of it with the noncollagenous carboxyl terminus (NC1) of vertebrate type IV basement membrane collagens (Blumberg, B., MacKrell, A. J., Olson, P. F., Kurkinen, M., Monson, J. M., Natzle, J. E., and Fessler, J. H. (1987) J. Biol. Chem. 262, 5947-5950). In the electron microscope each molecule appears as a thread with a knob at one end, which contains the carboxyl peptide domains. The variation of flexibility of the thread was mapped along its length. Pulse-chase labeling of cell cultures showed that these molecules associate into disulfide-linked dimers and higher oligomers that can be partly separated by velocity sedimentation and are resolved by sodium dodecyl sulfate-agarose gel electrophoresis. Dimers and higher oligomers formed by overlap of the amino ends of molecules were found. Mild pepsin digestion of Drosophila embryos and larvae solubilized the corresponding disulfide-linked collagen molecules, and Staphylococcus aureus V8 protease peptide maps showed the identity of the collagen derived from animals and from cell cultures. Individual, native molecules have a sedimentation coefficient s20,w = 4.1 S, the dichroic spectrum and amino acid composition of a collagen, and a Tm = 31 degrees C. Positive in situ hybridization with a specific probe for this collagen began 6-8 h after egg laying and showed message in the locations of embryos and larvae which reacted with the antibodies. This included some prominent individual cells in the hemolymph.

Anchoring fibrils contain the carboxyl-terminal globular domain of type VII procollagen, but lack the amino-terminal globular domain.

Type VII procollagen has been characterized as a product of epithelial cell lines. As secreted, it contains a large triple-helical domain terminated by a multi-globular-domained carboxyl terminus (NC-1), and a smaller amino-terminal globule (NC-2). The triple helix and the NC-1 domain have previously been identified in anchoring fibril-containing tissues by biochemical and immunochemical means, leading to the conclusion that type VII collagen is a major component of anchoring fibrils. In order to better characterize the tissue form of type VII collagen, we have produced a panel of monoclonal antibodies which recognize the NC-1 domain. Peptide mapping of these epitopes indicate that they are independent and span approximately 125,000 kDa of the total 150,000 kDa of each alpha chain contained in NC-1. All these antibodies elicit immunofluorescent staining of the basement membrane zone in tissues. Type VII collagen has been extracted from tissues. As previously reported, it is smaller than type VII procollagen, (Woodley, D. T., Burgeson, R. E., Lunstrum, G. P., Bruckner-Tuderman, L., and Briggaman, R. A., submitted for publication), and we now find that it predominantly occurs as a dimer. Following clostridial collagenase digestion, intact NC-1 has been recognized, indicating that the difference in apparent Mr between the tissue form of the molecule and type VII procollagen results from modification of the amino terminus. The size of the amino-terminal globule has been determined to be between approximately 96 and 102 kDa. Rotary shadowing analyses of extracted molecules indicate that dimeric molecules contain the NC-1 domain, but are missing intact NC-2. We propose that the tissue form monomer, Mr = 960,000, be referred to as "type VII collagen." These studies strongly suggest that anchoring fibrils contain dimeric molecules with intact NC-1 domains. The data also support the previous suggestion that the NC-2 domain is involved in the formation of disulfide bond-stabilized type VII collagen dimers, and is subsequently removed by physiological proteolytic processing.