Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Undulin, an extracellular matrix glycoprotein associated with collagen fibrils

Undulin, a novel noncollagenous extracellular matrix protein, was isolated from skin and placenta. In polyacrylamide gels most of the unreduced protein migrates with Mr above 1,000,000 yielding bands A (Mr 270,000), B1 (Mr 190,000), and B2 (Mr 180,000) after reduction. Undulin is biochemically and immunochemically distinct from other previously characterized large matrix glycoproteins. Immunoblotting using monoclonal antibodies suggests that bands A and B are closely related. Electron microscopy reveals undulin as structures consisting of an approximately 80-nm-long-tail with a nodule on one end and with one or two shorter arms on the other. Ultrastructurally immunolabeled undulin is found mainly between densely packed mature collagen fibrils. Indirect immunofluorescence shows bundles of uniform wavy fibers in dense connective tissues superimposable on a subpopulation of type I collagen structures. This suggests that undulin serves a specific yet unknown function in the supramolecular organization of collagen fibrils in soft tissues.     

The effect of catecholamines and adenosine on the induction of morphological alterations and depigmentation of newt iris epithelial cells in vitro

The J1 glycoproteins have been shown to mediate neuron-astrocyte adhesion and appear in the nervous system as four species of Mr 160,000 (J1-160), 180,000 (J1-180), 200,000 (J1-200), and 220,000 (J1-220), respectively. Tenascin is a disulfide-linked oligomeric, extracellular matrix glycoprotein of subunit Mr 170,000, 190,000, 200,000, and 220,000, which has been proposed to promote epithelial cell proliferation. In view of the structural similarities of the molecules we have used immunohistochemical and immunochemical techniques to compare them. Immunohistochemically, polyclonal J1 and tenascin antibodies yielded identical staining patterns in non-nervous-system tissues, and staining could be completely blocked by preincubating the sera with purified tenascin. In the central nervous system all structures expressing tenascin immunoreactivity were also recognized by J1 antibodies. However, not all J1-positive structures were also tenascin-positive, indicating that J1 antibodies recognized additional epitopes not present on tenascin. Western-blot experiments performed with affinity-purified polyclonal J1 antibodies showed that J1 glycoproteins can be subdivided into two separate pairs, J1-160/180 and J1-200/220, which share a small degree of homology. Western-blot experiments and sequential immunoprecipitations on biosynthetically [35S]methionine- or 125I-radiolabeled J1 glycoproteins carried out with polyclonal J1 and tenascin antibodies demonstrated that J1-200/220 is immunochemically indistinguishable from tenascin. These observations suggest that one set of extracellular glycoproteins is associated with processes as different as neural histogenesis and carcinogenesis of mammary glands.     

Human fibronectin and MMP-2 collagen binding domains compete for collagen binding sites and modify cellular activation of MMP-2.

The region of fibronectin (FN) surrounding the two type II modules of FN binds type I collagen. However, little is known about interactions of this collagen binding domain with other collagen types or extracellular matrix molecules. Among several expressed recombinant (r) human FN fragments from the collagen binding region of FN, only rI6-I7, which included the two type II modules and both flanking type I modules, bound any of several tested collagens. The rI6-I7 interacted specifically with both native and denatured forms of types I and III collagen as well as denatured types II, IV, V and X collagen with apparent K(d) values of 0.2-3.7 x 10(-7) M. Reduction with DTT disrupted the binding to gelatin verifying the functional requirement for intact disulfide bonds. The FN fragments showed a weak, but not physiologically important, binding to heparin, and did not bind elastin or laminin. The broad, but selective range of ligand interactions by rI6-I7 mirrored our prior observations for the collagen binding domain (rCBD) from matrix metalloproteinase-2 (MMP-2) [J. Biol. Chem. 270 (1995) 11555]. Subsequent experiments showed competition between rI6-I7 and rCBD for binding to gelatin indicating that their binding sites on this extracellular matrix molecule are identical or closely positioned. Two collagen binding domain fragments supported cell attachment by a beta1-integrin-dependent mechanism although neither protein contains an Arg-Gly-Asp recognition sequence. Furthermore, activation of MMP-2 and MMP-9 was greatly reduced for HT1080 fibrosarcoma cells cultured on either of the fibronectin fragments compared to full-length FN. These observations imply that the biological activities of FN in the extracellular matrix may involve interactions with a broad range of collagen types, and that exposure to pathologically-generated FN fragments may substantially alter cell behavior and regulation.     

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.     

The human fibroblast class II extracellular matrix receptor mediates platelet adhesion to collagen and is identical to the platelet glycoprotein Ia-IIa complex

A monoclonal antibody, P1H5, to the human fibroblast class II extracellular matrix receptor (ECMR II) specifically inhibits human fibroblast adhesion to collagen and immunoprecipitates a cell surface receptor containing an alpha and beta subunit of approximately 140 kilodaltons each (Wayner, E. A., and Carter, W. G. (1987) J. Cell Biol. 105, 1873-1884). We report here that P1H5 also specifically inhibits adhesion of unactivated human platelets to type I and III collagens, but not to fibronectin. Immunoprecipitation of the class II ECMR from Triton X-100 detergent lysates of platelets, after cell surface iodination, identified the platelet collagen receptor. Peptide mapping confirmed that the II alpha and II beta subunits immunoprecipitated from platelets are structurally homologous with those derived from fibroblasts. The platelet ECMR II alpha and -beta subunits comigrate with platelet membrane glycoproteins Ia and IIa, respectively, on two-dimensional nonreduced-reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. These results indicate that platelet and fibroblast adhesion to collagen are both mediated by a similar receptor and that the alpha and beta subunits of this receptor are identical to platelet membrane glycoproteins Ia and IIa, respectively. Although glycoprotein Ia has been previously implicated as a collagen binding protein, our results are the first direct evidence that platelet glycoprotein Ia is associated with glycoprotein IIa in a heterodimer complex and that this complex, by mediating platelet attachment, is an actual receptor for platelet adhesion to collagen.     

Cartilage oligomeric matrix protein interacts with type IX collagen, and disruptions to these interactions identify a pathogenetic mechanism in a bone dysplasia family

Cartilage oligomeric matrix protein (COMP) and type IX collagen are key structural components of the cartilage extracellular matrix and have important roles in tissue development and homeostasis. Mutations in the genes encoding these glycoproteins result in two related human bone dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia, which together comprise a "bone dysplasia family." It has been proposed that these diseases have a similar pathophysiology, which is highlighted by the fact that mutations in either the COMP or the type IX collagen genes produce multiple epiphyseal dysplasia, suggesting that their gene products interact. To investigate the interactions between COMP and type IX collagen, we have used rotary shadowing electron microscopy and real time biomolecular (BIAcore) analysis. Analysis of COMP-type IX collagen complexes demonstrated that COMP interacts with type IX collagen through the noncollagenous domains of type IX collagen and the C-terminal domain of COMP. Furthermore, peptide mapping identified a putative collagen-binding site that is associated with known human mutations. These data provide evidence that disruptions to COMP-type IX collagen interactions define a pathogenetic mechanism in a bone dysplasia family.     

Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.     

J1-160 and J1-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion

The glia-derived J1 extracellular matrix glycoproteins have been referred to as J1-160/J1-180 (the developmentally late appearing lower molecular weight group) and J1-200/J1-220 (the developmentally early appearing higher molecular group immunochemically related to tenascin). Members of the two groups show distinct cross-reactivities. To characterize the structural and functional differences between these J1 glycoproteins, two monoclonal antibodies were generated which recognize only the members of the lower molecular weight group. The two antibodies detect immunochemical similarities among the members of the lower molecular weight group, but do not react with J1/tenascin. J1-160 and J1-180 are specifically expressed by differentiated oligodendrocytes in culture and by myelin of the central nervous system and have not been found in the peripheral nervous system nor in any other organ of the adult mice tested. Electron microscopic examination of rotary-shadowed J1-160 and J1-180 reveals, respectively, dimeric and trimeric (tribrachion) kink-armed rodlike structures, which are linked by disulfide bridges. J1-160/J1-180 are nonpermissive substrates for the attachment and spreading of early postnatal small cerebellar neurons, astrocytes, and fibroblasts. In a mixture with laminin, J1- 160/J1-180 are nonpermissive substrates for neurons, but not for astrocytes or fibroblasts. The repulsive effect toward neurons can be neutralized by one of the monoclonal antibodies, but not by the other. These observations are discussed in the context of cell interactions during regeneration in the mammalian nervous system.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Independent deposition of collagen types II and IX at epithelial-mesenchymal interfaces

Previous studies have demonstrated the presence of type II collagen (in mature chickens predominantly a 'cartilage-specific' collagen) in a variety of embryonic extracellular matrices that separate epithelia from mesenchyme. In an immunohistochemical study using collagen type-specific monoclonal antibodies, we asked whether type IX collagen, another 'cartilage-specific' collagen, is coexpressed along with type II at such interfaces. We confirmed that, in the matrix underlying a variety of cranial ectodermal derivatives and along the ventrolateral surfaces of neuroepithelia, type II collagen is codistributed with collagen types I and IV. Type IX collagen, however, was undetectable at those sites. We observed immunoreactivity for type IX collagen only within the notochordal sheath, where it first appeared at a later stage than did collagen types I and II. We also observed type II collagen (without type IX) beneath the dorsolateral ectoderm at stage 16; this correlates with the period during which limb ectoderm has been reported to induce the mesoderm to become chondrogenic. Finally, in older hind limbs we observed subepithelial type II collagen that was not associated with subsequent chondrogenesis, but appeared to parallel the formation of feathers and scales in the developing limb. These observations suggest that the deposition of collagen types II and IX into interfacial matrices is regulated independently, and that induction of mesenchymal chondrogenesis by such matrices does not involve type IX collagen. Subepithelial type IX collagen deposition, on the other hand, correlates with the assembly of a thick multilaminar fibrillar matrix, as present in the notochordal sheath and, as shown previously, in the corneal primary stroma.     

Selective binding of collagen subtypes by integrin alpha 1I, alpha 2I, and alpha 10I domains

Four integrins, namely alpha(1)beta(1), alpha(2)beta(1), alpha(10)beta(1), and alpha(11)beta(1), form a special subclass of cell adhesion receptors. They are all collagen receptors, and they recognize their ligands with an inserted domain (I domain) in their alpha subunit. We have produced the human integrin alpha(10)I domain as a recombinant protein to reveal its ligand binding specificity. In general, alpha(10)I did recognize collagen types I-VI and laminin-1 in a Mg(2+)-dependent manner, whereas its binding to tenascin was only slightly better than to albumin. When alpha(10)I was tested together with the alpha(1)I and alpha(2)I domains, all three I domains seemed to have their own collagen binding preferences. The integrin alpha(2)I domain bound much better to fibrillar collagens (I-III) than to basement membrane type IV collagen or to beaded filament-forming type VI collagen. Integrin alpha(1)I had the opposite binding pattern. The integrin alpha(10)I domain was similar to the alpha(1)I domain in that it bound very well to collagen types IV and VI. Based on the previously published atomic structures of the alpha(1)I and alpha(2)I domains, we modeled the structure of the alpha(10)I domain. The comparison of the three I domains revealed similarities and differences that could potentially explain their functional differences. Mutations were introduced into the alphaI domains, and their binding to types I, IV, and VI collagen was tested. In the alpha(2)I domain, Asp-219 is one of the amino acids previously suggested to interact directly with type I collagen. The corresponding amino acid in both the alpha(1)I and alpha(10)I domains is oppositely charged (Arg-218). The mutation D219R in the alpha(2)I domain changed the ligand binding pattern to resemble that of the alpha(1)I and alpha(10)I domains and, vice versa, the R218D mutation in the alpha(1)I and alpha(10)I domains created an alpha(2)I domain-like ligand binding pattern. Thus, all three collagen receptors appear to differ in their ability to recognize distinct collagen subtypes. The relatively small structural differences on their collagen binding surfaces may explain the functional specifics.     

Characterization of binding properties of the myelin-associated glycoprotein to extracellular matrix constituents.

The myelin-associated glycoprotein (MAG) can be obtained from adult mouse brain from detergent-lysates of a crude membrane fraction as a 96-100 kd form (detergent solubilized MAG), and from 100,000 g supernatants of homogenates as a 90-96 kd form (soluble MAG). The soluble form distributes into the Triton X-114-poor aqueous phase, while detergent-solubilized MAG predominantly enters the Triton X-114-rich phase. Both molecular forms bind to heparin in hypo- and isotonic buffers. Soluble MAG binds to several collagens (type G, I, II, III, IV, V, VI, IX) with a kd of 5.7 X 10(-8) M for collagen type IX and 2.0 X 10(-7) for collagen type IV. Binding of 125I-labeled MAG to collagen G can be completely inhibited by unlabeled MAG and collagen G, but not by heat-denatured collagen. MAG does not bind to itself, laminin, fibronectin, or the neural cell adhesion molecules L1 and N-CAM. Binding of MAG to collagen G is most effectively blocked by a high molecular weight dextran sulfate, heparan sulfate and heparin, with chondroitin sulfate and a low molecular weight dextran sulfate being less potent blockers. These findings are in agreement with previous observations on the localization of MAG in basal lamina and interstitial collagens of the sciatic nerve in situ.     

Mapping the collagen-binding site in the von Willebrand factor-A3 domain

The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion to collagen at sites of vascular damage. The binding site for collagen types I and III is located in the VWF-A3 domain. Recently, we showed that His(1023), located near the edge between the "front" and "bottom" faces of A3, is critical for collagen binding (Romijn, R. A., Bouma, B., Wuyster, W., Gros, P., Kroon, J., Sixma, J. J., and Huizinga, E. G. (2001) J. Biol. Chem. 276, 9985-9991). To map the binding site in detail, we introduced 22 point mutations in the front and bottom faces of A3. The mutants were expressed as multimeric VWF, and binding to collagen type III was evaluated in a solid-state binding assay and by surface plasmon resonance. Mutation of residues Asp(979), Ser(1020), and His(1023) nearly abolished collagen binding, whereas mutation of residues Ile(975), Thr(977), Val(997), and Glu(1001) reduced binding affinity about 10-fold. Together, these residues define a flat and rather hydrophobic collagen-binding site located at the front face of the A3 domain. The collagen-binding site of VWF-A3 is distinctly different from that of the homologous integrin alpha(2) I domain, which has a hydrophilic binding site located at the top face of the domain. Based on the surface characteristics of the collagen-binding site of A3, we propose that it interacts with collagen sequences containing positively charged and hydrophobic residues. Docking of a collagen triple helix on the binding site suggests a range of possible engagements and predicts that at most eight consecutive residues in a collagen triple helix interact with A3.     

Sites of stromelysin cleavage in collagen types II, IX, X, and XI of cartilage

Human recombinant stromelysin-1 was shown to cleave four types of collagen (types II, IX, X, and XI) prepared from bovine and rat cartilages at specific sites. Stromelysin-1 cleaved salt-soluble native molecules of type IX collagen into two main triple-helical fragments, COL1 and COL2,3. Protein microsequencing identified the exact cleavage sites in the NC2 domain of all three chains, alpha 1(IX), alpha 2(IX), and alpha 3(IX). Stromelysin-1 also acted as a "telopeptidase," in that it efficiently clipped intact molecules of types II and XI collagens at sites just inside their terminal cross-linking hydroxylysine residues. Native molecules of type X collagen were cleaved by stromelysin-1 within their triple helical domains at a COOH-terminal site that reduced the alpha 1(X) chain size by 10 kDa. These findings suggest an important role for stromelysin in the turnover and remodeling of the collagenous matrix of cartilage both normally and in degenerative joint disease.     

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

A soluble form of the neural cell adhesion molecule (N-CAM) was obtained from 100,000-g supernatants of crude brain membrane fractions by incubation for 2 h at 37 degrees C. The isolated N-CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N-CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N-CAM 110 bound to different types of collagen (collagen types I-VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fibronectin). (b) Binding of N-CAM 110 to heat-denatured collagens was absent or substantially reduced. (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3-2.0 X 10(-9) M, depending on the collagen type and buffer conditions). Binding of N-CAM 110 to collagens could be prevented in a concentration-dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N-CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell-cell adhesion, N-CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo.     

Interaction of astrochondrin with extracellular matrix components and its involvement in astrocyte process formation and cerebellar granule cell migration

We have recently characterized a chondroitin sulfate proteoglycan from the murine central nervous system which is expressed by astrocytes in vitro and carries the L2/HNK-1 and L5 carbohydrate structures. In the present study, we provide evidence that its three core proteins of different size are similar in their proteolytic peptide maps and thus designate this group of structurally related molecules astrochondrin. During development, astrochondrin and the L5 carbohydrate were hardly detectable in the brain of 14-d-old mouse embryos by Western blot analysis. Expression of astrochondrin and the L5 epitope was highest at postnatal day 8, the peak of cerebellar granule cell migration and Bergmann glial process formation, and decreased to weakly detectable levels in the adult. Immunocytochemical localization of astrochondrin in the cerebellar cortex of 6-d-old mice showed association of immunoreactivity with the cell surface of astrocytes, including Bergmann glial processes and astrocytes in the internal granular layer or prospective white matter. Endfeet of astrocytes contacting the basal lamina of endothelial and meningeal cells and contact sites between Bergmann glial processes and granule cells also showed detectable levels of astrochondrin. Furthermore, granule cell axons in the molecular layer were astrochondrin immunoreactive. In the adult, astrochondrin immunoreactivity was weakly present in the internal granular layer and white matter. Both Fab fragments of polyclonal antibodies to astrochondrin and monovalent fragments of the L5 monoclonal antibody reduced the formation of processes of mature GFAP- positive astrocytes on laminin and collagen type IV, but not on fibronectin as substrata. Interestingly, the initial attachment of astrocytic cell bodies was not disturbed by these antibodies. Antibodies to astrochondrin also reduced the migration of granule cells in the early postnatal mouse cerebellar cortex. In a solid phase radioligand binding assay, astrochondrin was shown to bind to the extracellular matrix components laminin and collagen type IV, being enhanced in the presence of Ca2+, but not to fibronectin, J1/tenascin or other neural recognition molecules. Furthermore, astrochondrin interacted with collagen types III and V, less strongly with collagen types I, II, and IX, but not with collagen type VI. The interaction of astrochondrin with collagen types III and V was saturable and susceptible to increasing ionic strength, and could be competed by chondroitin sulfate, heparin, and dextran sulfate, but not by hyaluronic acid, glucose-6-phosphate, or neuraminic acid.(ABSTRACT TRUNCATED AT 400 WORDS)