Membrane-associated proteoglycans produced by Sertoli cells are not randomly distributed on the cell surface.

Sertoli cells in culture synthesize two membrane-associated proteoglycans (PGs) containing as glycosaminoglycan (GAG) moieties either chondroitin sulfate (CS) or CS and heparan sulfate (HS); the latter PG is, therefore, referred to as the mixed PG. To determine if these PGs are randomly distributed on the cell surface, Sertoli cell monolayers were treated with chondroitinase ABC, and then the remaining PGs were analyzed by DEAE-Sephacel chromatography. The results obtained with Sertoli cell monolayers show that the CS of the mixed PG is degraded by chondroitinase, while the CS-PG is not degraded. In contrast, chondroitinase treatment of Sertoli cells in suspension shows that both the mixed PG and the CS-PG are degraded. From this, it is inferred that the mixed PG is apically oriented and the CS-PG is basolaterally oriented. Studies of the adhesion of germ cells to Sertoli cell monolayers give further support to the apical location of the mixed PG and suggest that its HS moiety is involved in the attachment of germ cells to Sertoli cells.     

Obligatory requirement of sulfation for P-selectin binding to human salivary gland carcinoma Acc-M cells and breast carcinoma ZR-75-30 cells

Stimulated endothelial cells and activated platelets express P-selectin, which reacts with P-selectin glycoprotein ligand-1 (PSGL-1) for leukocyte rolling on the stimulated endothelial cells and heterotypic aggregation of the activated platelets on leukocytes. P-selectin also binds to several cancer cells in vitro and promotes the growth and metastasis of human colon carcinoma in vivo. The P-selectin/PSGL-1 interaction requires tyrosine sulfation. However, it is unknown whether sulfation is necessary for P-selectin binding to somatic cancer cells. In this study, we show that P-selectin mediated adhesion of Acc-M cells, a cell line derived from a human adenoid cystic carcinoma of salivary gland. These cells had a moderate expression of heparan sulfate-like proteoglycans, but had no detectable expressions of PSGL-1, CD24, Lewis(x), and sialyl Lewis(x). Treatment with sodium chlorate (a sulfation biosynthesis inhibitor), but not 4-methylumbelliferyl-beta-D-xyloside (a proteoglycan biosynthesis inhibitor) or heparinases, reduced adhesion of these cells to P-selectin. Sodium chlorate also inhibited the P-selectin precipitation of the 160-, 54-, and 36-kDa molecules from the cell surface of Acc-M cells. Furthermore, P-selectin could bind to human breast carcinoma ZR-75-30 cells in a sulfation-dependent manner. Our results thus indicate that sulfation is essential for adhesion of nonblood-borne, epithelial-like human cancer cells to P-selectin.     

Tyrosine sulfation site is located in the C-terminal fibrin-binding domain in secreted fibronectin from rat embryo fibroblasts, line 3Y1

Tryptic fragments of [35S]sulfate-labeled 3Y1 secreted fibronectin were fractionated by hydroxylapatite column chromatography and examined using sodium dodecyl sulfate gel electrophoresis, followed by autoradiography. Radioactive bands containing tyrosine-O-[35S]sulfate were detected at 17- and 40-kDa positions under reducing conditions. Under nonreducing conditions, the 17-kDa band was no longer present and new bands at 57- and 80-kDa positions appeared, indicating a disulfide linkage between the two smaller fragments in the native state. These fragments exhibited binding affinity toward fibrin and could be immunoprecipitated by the monoclonal antifibronectin Fib-2 domain antibody. These results suggested that the tyrosine sulfation site in 3Y1 secreted fibronectin is located in the C-terminal fibrin-binding (Fib-2) domain, being within 17 kDa of the C-terminus.     

Concurrent reduction in the sulfation of heparan sulfate and basement membrane assembly in a cell model system

Basement membranes (BMs) are specialized extracellular matrices that have important roles in cell attachment, migration, growth and differentiation. The murine teratocarcinoma cell line, M1536-B3, has been shown to produce a model BM composed of laminin, entactin and heparan sulfate proteoglycans but lacking collagen. Therefore, M1536-B3 cells are an excellent model system in which to study the role of non-collagenous components in BM assembly. We have used these cells to test for a requirement of mature heparan sulfate (HS) chains in BM assembly. Growth of M1536-B3 cells in the presence of chlorate, an inhibitor of activated sulfate synthesis, resulted in a dose-dependent decrease in the sulfation of glycosaminoglycans and reduction in the charge density of the isolated HS. The undersulfated HS from chlorate-treated cells had a decreased binding capacity for laminin when compared with control HS. Concurrent with these changes in sulfation, chlorate treatment of M1536-B3 cells resulted in the failure of BM assembly, which was restored upon removal of the chlorate from the growth medium. These results were not due to major alterations in cell attachment, spreading, growth, protein synthesis, or to an inability of the cells to synthesize and secrete laminin. These data suggest that the sulfation of HS and its subsequent ability to interact with other BM components play major roles in the assembly and structure of BMs.     

Inhibition of tyrosine sulfation in the trans-Golgi retards the transport of a constitutively secreted protein to the cell surface

The effect of tyrosine sulfation on the transport of a constitutively secreted protein, yolk protein 2 (YP2) of Drosophila melanogaster, to the cell surface was investigated after expression of YP2 in mouse fibroblasts. Inhibition of YP2 sulfation was achieved by two distinct approaches. First, the single site of sulfation in YP2, tyrosine 172, was changed to phenylalanine by oligonucleotide-directed mutagenesis. Second, L cell clones stably expressing YP2 were treated with chlorate, a reversible inhibitor of sulfation. Pulse-chase experiments with transfected L cell clones showed that the half-time of transport from the rough endoplasmic reticulum to the cell surface of the unsulfated mutant YP2 and the unsulfated wild-type YP2 produced in the presence of chlorate was 15-18 min slower than that of the sulfated wild-type YP2. Control experiments indicated (a) that the tyrosine to phenylalanine change itself did not affect YP2 transport, (b) that the retardation of YP2 transport by chlorate occurred only with sulfatable but not with unsulfatable YP2, (c) that the transport difference between wild-type and mutant YP2 was not due to the level of YP2 expression, and (d) that transport of the endogenous secretory protein fibronectin was the same in L cell clones expressing wild-type and mutant YP2. Since the half-time of transport of wild-type YP2 from the intracellular site of sulfation, the trans-Golgi, to the cell surface was found to be 10 min, the 15-18-min retardation seen upon inhibition of tyrosine sulfation reflected a two- to threefold increase in the half-time of trans-Golgi to cell surface transport, which was most probably caused by an increased residence time of unsulfated YP2 in the trans-Golgi. The results demonstrate a role of tyrosine sulfation in the intracellular transport of a constitutively secreted protein.     

Chemical inhibition of sulfation accelerates neural differentiation of mouse embryonic stem cells and human induced pluripotent stem cells

Pluripotency of embryonic stem cells (ESCs) is maintained by the balancing of several signaling pathways, such as Wnt, BMP, and FGF, and differentiation of ESCs into a specific lineage is induced by the disruption of this balance. Sulfated glycans are considered to play important roles in lineage choice of ESC differentiation by regulating several signalings. We examined whether reduction of sulfation by treatment with the chemical inhibitor chlorate can affect differentiation of ESCs. Chlorate treatment inhibited mesodermal differentiation of mouse ESCs, and then induced ectodermal differentiation and accelerated further neural differentiation. This could be explained by the finding that several signaling pathways involved in the induction of mesodermal differentiation (Wnt, BMP, and FGF) or inhibition of neural differentiation (Wnt and BMP) were inhibited in chlorate-treated embryoid bodies, presumably due to reduced sulfation on heparan sulfate and chondroitin sulfate. Furthermore, neural differentiation of human induced pluripotent stem cells (hiPSCs) was also accelerated by chlorate treatment. We propose that chlorate could be used to induce efficient neural differentiation of hiPSCs instead of specific signaling inhibitors, such as Noggin.     

Under-sulfation by PAPS synthetase inhibition modulates the expression of ECM molecules during chondrogenesis

Sulfation of proteoglycans is an important post-translational modification in chondrocytes. We previously found that 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2 levels increased more than 10-fold during mesenchymal cell chondrogenesis. Given that PAPS is the sole sulfur donor, and is produced only by PAPS synthetase in all cells, increased expression of PAPS synthetase-2 should be a prerequisite for increased sulfation activity of chondrocytes. We found that sodium chlorate, a specific inhibitor of PAPS synthetase, inhibited proteoglycan sulfation during chondrogenesis. In contrast, sodium chlorate unexpectedly induced early expression of type II collagen and increased the number of cartilage nodules during chondrogenesis. Inhibition of sulfation also accelerated the down-regulation of N-cadherin and fibronectin during chondrogenesis. These findings suggest that sulfation has an important regulatory role in coordinating the timely expression of extracellular matrix molecules during chondrogenesis, and that under-sulfation may cause the breakdown of this coordination, leading to premature chondrogenesis.     

Role of sulfation in the processing of gastric mucins.

The role of sulfation in the processing of mucus glycoprotein in gastric mucosa was investigated. Rat gastric mucosal segments were incubated in MEM at various medium sulfate concentrations in the presence of [35S]Na2SO4, [3H]glucosamine and [3H]proline, with and without chlorate an inhibitor of PAPS formation. The results revealed that the mucin sulfation attained maximum at 300 microM medium sulfate concentration. Introduction of chlorate into the incubation medium, while having no effect on the protein synthesis as evidenced by [3H]proline incorporation, caused at its optimal concentration of 2 mM a 90% decrease in mucin sulfation and a 40% drop in mucin glycosylation. Evaluation of mucin molecular forms distribution indicated the predominance of the high molecular mucin form in the intracellular fraction and the low molecular mucin from in the extracellular fraction. Increase in medium sulfate caused an increase in the high molecular weight mucin form in both fractions, and this effect was inhibited by chlorate. Also, higher medium sulfate concentrations led to a higher degree of sulfation in the high molecular weight mucin form, the effect of which was inhibited by chlorate. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and is required for mucin polymer formation. Hence, the disturbances in mucin sulfation process could be detrimental to the maintenance of gastric mucus coat integrity.     

Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.     

Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System

Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.     

Evidence for the Binding of Ng-CAM to Laminin

Ng-CAM is a cell adhesion molecule mediating neuron-glia and neuron-neuron adhesion via different binding mechanisms. While its binding can be homophilic as demonstrated by the self-aggregation of Ng-CAM coated beads (Covaspheres), Ng-CAM has also been shown to bind to glia by a heterophilic mechanism. In the present study, we found that the extent of Ng-CAM Covasphere aggregation was strongly diminished in the presence of the extracellular matrix glycoprotein laminin. When proteolytic fragments of laminin were tested, the P1' fragment (obtained from the short arms by pepsin treatment) was found to inhibit aggregation of Ng-CAM-Covaspheres while the elastase fragments E3 and E8 (from the long arm) were ineffective. To provide other means of analyzing interactions between laminin and Ng-CAM, the two proteins were covalently linked to differently fluorescing Covaspheres and tested for coaggregation. Laminin-Covaspheres coaggregated with Ng-CAM-Covaspheres, and this binding was inhibited both by anti-Ng-CAM and by anti-laminin antibodies. Covaspheres coated with other proteins including BSA and fibronectin did not coaggregate with Ng-CAM-Covaspheres. Moreover, using a solid phase binding assay, we found that ¹²⁵I-labeled Ng-CAM bound to laminin and to Ng-CAM but not to fibronectin. The results suggest that regions in the short arms of laminin can bind to Ng-CAM. To test whether Ng-CAM present on neurons could be involved in binding to laminin, adhesion of neurons to substrates coated with various proteins was tested in the presence of specific antibodies. Anti-Ng-CAM Fab' fragments inhibited neuronal binding to laminin but not binding to fibronectin. The combined results open the possibility that Ng-CAM on the surface of neurons may mediate binding to laminin in vivo, and that interactions with laminin can modulate homophilic Ng-CAM binding.     

Distinct Structural Requirements for Interaction of the Integrins α5β1, αvβ5, and αvβ6 with the Central Cell Binding Domain in Fibronectin

At least 10 different members of the integrin family have been reported to bind to fibronectin, and eight of these interact with the arginine-glycine-aspartic acid (RGD) site in the tenth type III repeat. However, studies utilizing recombinant fibronectin fragments have shown that for three of these, α5β1, αIIbβ3, and αvβ3, the structural requirements for binding to fibronectin differ. In the present study. we report that two additional integrins, αvβ6. and αvβ5 also demonstrate unique requirements for interaction with recombinant fibronectin fragments. αvβ5, like αvβ3, can support cell adhesion to the RGD-containing tenth repeat alone, and does not require the presence of a synergy site in the adjacent ninth repeat. In the cells used in this study. αvβ5 only minimally supported adhesion to intact fibronectin. but did support adhesion to fragments composed of the eighth, ninth and tenth repeats or the tenth repeat. alone. Mutant fragments in which the eighth and tenth repeats were adjacent to one another enhanced adhesion mediated by αvβ5, as well as adhesion mediated by αvβ6. αvβ5 and αvβ6-mediated adhesion to all fibronectin fragments required interaction with the RGD site, as inferred by inhibition of adhesion with an RGD-containing peptide. These data suggest that each integrin that interacts with the RGD site in fibronectin has unique structural requirements for this interaction.     

Effect of chlorate on the sulfation of lipoprotein lipase and heparan sulfate proteoglycans. Sulfation of heparan sulfate proteoglycans affects lipoprotein lipase degradation

In avian-cultured adipocytes 76% of the newly synthesized lipoprotein lipase is degraded before release into the medium (Cupp, M., Bensadoun, A., and Melford, K. (1987) J. Biol. Chem. 262, 6383-6388). The same group (Cisar, L. A., Hoogewerf, A. J., Cupp, M., Rapport, C. A., and Bensadoun, A. (1989) J. Biol. Chem. 264, 1767-1774) has proposed that the interaction of lipoprotein lipase with a class of cell surface heparan sulfate proteoglycans is necessary for degradation to occur. To test further this hypothesis, the binding capacity of the plasma membrane for the lipase was decreased by inhibiting the sulfation of glycosaminoglycans with sodium chlorate, an inhibitor of sulfate adenyltransferase. Chlorate decreased sulfate incorporation into trypsin-releasable heparan sulfate proteoglycans to 20% of control levels. The amount of uronic acid in the trypsin-releasable heparan sulfate proteoglycans remained constant. Therefore, chlorate decreased sulfation density on heparan sulfate chains by approximately 5-fold. In the same fractions, chlorate increased the median heparan sulfate Mr measured on Sephacryl S-300. Chlorate decreased the maximum binding of 125I-lipoprotein lipase to adipocytes by 4-fold, but no significant effects on the affinity constants were observed. Chlorate increased lipoprotein lipase secretion in a dose-dependent relationship up to 30 mM. Utilizing a pulse-chase protocol, it was shown that lipase synthesis in control and chlorate-treated cells was not significantly different and that the increased secretion could be accounted for by a decreased lipoprotein lipase degradation rate. In control cells 77 +/- 11% of the synthesized enzyme was degraded whereas in chlorate-treated cells degradation was reduced to 42 +/- 9% of the synthesized amount. The present study shows that decreased sulfation of heparan sulfate proteoglycans decreases the maximum binding of the lipase for the adipocyte cell surface. Consistent with the model that binding of lipoprotein lipase to cell surface heparan sulfate is required for lipase degradation, degradation is reduced in chlorate-treated cultures. In this report it is also shown that chlorate inhibits lipoprotein lipase sulfation and that desulfation of the enzyme has no effect on its catalytic efficiency or on its binding to cultured adipocytes.     

Role of sulfation in post-translational processing of gastric mucins.

1. Gastric mucosal segments were incubated in MEM supplemented with various sulfate concentrations in the presence of [3H]glucosamine, [3H]proline and [35S]Na2SO4, with and without chlorate, an inhibitor of 3'-phosphoadenosine-5'-phosphosulfate formation. 2. Incorporation of glucosamine and sulfate depended upon the sulfate content of the medium and reached a maximum at 300 microM sulfate. Introduction of chlorate into the medium, while having no effect on protein synthesis as evidenced by proline incorporation, caused, at its optimal concentration of 2 mM, a 90% decrease in mucin sulfation and a 40% drop in glycosylation. 3. At low sulfate content in the medium and in the presence of chlorate, the incorporation of sulfate and glucosamine was mainly into the low molecular-weight form of mucin. An increase in sulfate in the medium caused an increase in the high molecular-weight form of mucin and in the extent of sulfation in its carbohydrate chain. 4. The results suggest that the sulfation process is an early event taking place at the stage of mucin subunit assembly and that sulfate availability is essential for the formation of the high molecular-weight mucin polymer.     

Heparan Sulfate Proteoglycans Play a Dual Role in Regulating Fibroblast Growth Factor-2 Mitogenic Activity in Human Breast Cancer Cells

The human breast cancer cell lines MCF-7 and MDA-MB-231 differ in their responsiveness to fibroblast growth factor-2 (FGF-2). This growth factor stimulates proliferation in well-differentiated MCF-7 cells, whereas the less well-differentiated MDA-MB-231 cells are insensitive to this molecule. To investigate the potential regulation of FGF-2 mitogenic activity by heparan sulfate proteoglycans (HSPG), we have treated human breast cancer cells by glycosaminoglycan degrading enzymes or a metabolic inhibitor of proteoglycan sulfation: sodium chlorate. The interaction between FGF-2 and proteoglycans was assayed by examining the binding of¹²⁵I-FGF-2 to breast cancer cell cultures as well as to cationic membranes loaded with HSPG. Using MCF-7 cells, we showed that heparinase treatment inhibited FGF-2 binding to HSPG and completely abolished FGF-2 induced growth; chlorate treatment of MCF-7 cells decreased FGF-2 binding to HSPG and cell responsiveness in a dose-dependent manner. This demonstrates a requirement of adequately sulfated HSPG for FGF-2 growth-promoting activity on MCF-7 cells. In highly invasive MDA-MB-231 cells which produce twice as much HSPG as MCF-7 cells and which are not normally responsive to exogenously added FGF-2, chlorate treatment decreased FGF-2 binding to HSPG and induced FGF-2 mitogenic effect. This chlorate effect was dose dependent and observed at concentrations of 10–30 mM;higher chlorate concentrations completely abolished the FGF-2 effect. This shows that the HSPG level of sulfation can also negatively regulate the biological activity of FGF-2. Taken together, these results demonstrate a crucial role for HSPG in both positive and negative control of FGF-2 mitogenic activity in breast cancer cell proliferation.     

Extracellular matrix is required for skeletal muscle differentiation but not myogenin expression

Skeletal muscle cells are a useful model for studying cell differentiation. Muscle cell differentiation is marked by myoblast proliferation followed by progressive fusion to form large multinucleated myotubes that synthesize muscle-specific proteins and contract spontaneously. The molecular analysis of myogenesis has advanced with the identification of several myogenic regulatory factors, including myod1, myd, and myogenin. These factors regulate each other's expression and that of muscle-specific proteins such as the acetylcholine receptor and acetylcholinesterase (AChE). In order to investigate the role of extracellular matrix (ECM) in myogenesis we have cultured myoblasts (C₂C₁₂) in the presence or absence of an exogenous ECM (Matrigel). In addition, we have induced differentiation of myoblasts in the presence or absence of Matrigel and/or chlorate, a specific inhibitor of proteoglycan sulfation. Our results indicated that the formation of fused myotubes and expression of AChE was stimulated by Matrigel. Treatment of myoblasts induced to differentiate with chlorate resulted in an inhibition of cell fusion and AChE activity. Chlorate treatment was also found to inhibit the deposition and assembly of ECM components such fibronectin and laminin. The expression of myogenin mRNA was observed when myoblasts were induced to differentiate, but was unaffected by the presence of Matrigel or by culture of the cells in the presence of chlorate. These results suggest that the expression of myogenin is independent of the presence of ECM, but that the presence of ECM is essential for the formation of myotubes and the expression of later muscle-specific gene products. © 1996 Wiley-Liss, Inc.     

Inhibition of binding of fibronectin to matrix assembly sites by anti- integrin (alpha 5 beta 1) antibodies

Fibroblasts have cell surface sites that mediate assembly of plasma and cellular fibronectin into the extracellular matrix. Cell adhesion to fibronectin can be mediated by the interaction of an integrin (alpha 5 beta 1) with the Arg-Gly-Asp-Ser (RGDS)-containing cell adhesion region of fibronectin. We have attempted to elucidate the role of the alpha 5 beta 1 fibronectin receptor in assembly of fibronectin in matrices. Rat monoclonal antibody mAb 13, which recognizes the integrin beta 1 subunit, completely blocked binding and matrix assembly of 125I-fibronectin as well as binding of the 125I-70-kD amino-terminal fragment of fibronectin (70 kD) to fibroblast cell layers. Fab fragments of the anti-beta 1 antibody were also inhibitory. Antibody mAb 16, which recognizes the integrin alpha 5 subunit, partially blocked binding of 125I-fibronectin and 125I-70-kD. When cell layers were coincubated with fluoresceinated fibronectin and either anti-beta 1 or anti-alpha 5, anti-beta 1 was a more effective inhibitor than anti-alpha 5 of binding of labeled fibronectin to the cell layer. Inhibition of 125I-fibronectin binding by anti-beta 1 IgG occurred within 20 min. Inhibition of 125I-fibronectin binding by anti-beta 1 Fab fragments or IgG could not be overcome with increasing concentrations of fibronectin, suggesting that anti-beta 1 and exogenous fibronectin may not compete for the same binding site. No beta 1-containing integrin bound to immobilized 70 kD. These data indicate that the beta 1 subunit plays an important role in binding and assembly of exogenous fibronectin, perhaps by participation in the organization, regeneration, or cycling of the assembly site rather than by a direct interaction with fibronectin.     

A second fibronectin-binding region is present in collagen alpha chains

The interactions of plasma fibronectin with alpha chains or cyanogen bromide fragments of collagen types I and II have been studied using a variety of techniques. Affinity chromatography of cyanogen bromide-cleaved type II collagen on immobilized fibronectin revealed the binding of cyanogen bromide fragment CB12 in addition to the previously characterized CB10. Using fluorescence polarization, we analyzed the interaction between the collagen peptides and fluorescein isothiocyanate-labeled 42-kDa gelatin-binding fragment of fibronectin in solution. Dissociation constants for the binding of CB10 and CB12 to the fibronectin fragment were calculated as 0.38 and 0.94 microM, respectively, indicating a lower affinity for the uncharacterized site. However, as with CB10, CB12 was able to compete effectively with the intact alpha chain for bindinng to fibronectin. Additionally, both CB10 and CB12 absorbed to tissue culture surfaces were each able to support fibronectin-dependent cell adhesion. Finally, the regions of alpha 2(I) homologous to CB12 and CB10 were found to be active in fibronectin binding, demonstrating the presence of two fibronectin-binding regions in this collagen chain.     

Fibronectin-plasma membrane interaction in the adhesion of hemopoietic cells

Many hemopoietic cell lines were examined for their ability to adhere to culture dishes coated with extracellular matrix proteins. Adhesion assay was performed with murine and human leukemic cell lines representative of different stages of differentiation along both erythroid and myeloid lineages. All the hemopoietic cell lines tested adhered to fibronectin but not to laminin, types I, III, and IV collagen, serum-spreading factor, and cartilage proteoglycans. In addition to immortalized cell lines, immature erythroid and myeloid mouse bone marrow cells adhered to fibronectin. To define the fibronectin region involved in hemopoietic cell adhesion, proteolytic fragments, monoclonal antibodies, and synthetic peptides were used. Among different fibronectin fragments tested, only a 110-kD polypeptide, corresponding to the fibroblast attachment domain, was active in promoting adhesion. Moreover, a monoclonal antibody to the cell binding site located within this domain prevented hemopoietic cell adhesion. Finally, the tetrapeptide Arg-Gly-Asp-Ser, which corresponds to the fibronectin sequence recognized by fibroblastic cells, specifically and competitively inhibited attachment of hemopoietic cells to this molecule. The cell surface molecule involved in the interaction of mouse hemopoietic cells with fibronectin was identified as a 145,000-D membrane glycoprotein by adhesion-blocking antibodies. This glycoprotein was found to be antigenically and functionally related to the GP135 membrane glycoprotein involved in the adhesion of fibroblasts to fibronectin (Giancotti, F. G., P. M. Comoglio, and G. Tarone, 1986, Exp. Cell Res., 163:47-62). On the basis of these data, we conclude that interaction of hemopoietic cells with fibronectin involves a specific fibronectin sequence and a 145,000-D cell surface glycoprotein. We speculate that this property might be relevant for the interaction of hemopoietic cells with the bone marrow stroma, which represents the natural site of hemopoiesis.