A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway

The ligand specificity of the human monocyte receptor that mediates phagocytosis of particulate activators of the human alternative complement pathway was defined by inhibiting the phagocytic response with glycans known to be present in zymosan. When monocytes in monolayers were preincubated with 100 micrograms/ml of beta-glucan and then incubated with 1.25 to 2.5 X 10(6) zymosan particles, the percentage of cells that exhibited phagocytosis was inhibited in a time-dependent manner; maximal inhibition occurred within 20 min of preincubation. beta-Glucan inhibited monocyte phagocytosis of zymosan and rabbit erythrocytes (Er) in a similar dose-dependent fashion and at 100 micrograms/ml reduced monocyte ingestion of 5 X 10(6)/ml zymosan and 2 X 10(8)/ml Er by 63 +/- 8% and 68 +/- 16% (mean +/- SD, n = 3), respectively. The other glycan constituent of zymosan, mannan, was less than 1% as active, and 10 mg/ml of mannan reduced the number of monocytes ingesting zymosan and Er by 56 +/- 12% and 26 +/- 11%, respectively. At concentrations as high as 500 micrograms/ml, beta-glucan had no effect on monocyte Fc, C3b, or fibronectin receptor-mediated functions. Enzymatic hydrolysis of beta-glucan and alpha-mannan with beta-glucosidase or beta-glucanase before their incubation with monocytes abrogated their inhibitory capacity, whereas hydrolysis with alpha-mannosidase or alpha-glucosidase did not. Neither of the two alpha-glucans tested (dextran T-70 and nigeran) affected monocyte ingestion of zymosan particles or sheep erythrocytes (Es) sensitized with rabbit 7S anti-Es (EsIgG) at concentrations as high as 2 mg/ml. In contrast, a number of beta-glucans were active against zymosan but not EsIgG ingestion with a 75% reduction in the number of monocytes ingesting zymosan occurring with 100 micrograms/ml laminarin, 500 micrograms/ml soluble pachyman, and 900 micrograms/ml of soluble pustulan. The galactan, agarose, either in suspensions at 2 mg/ml or in a soluble portion at 600 micrograms/ml failed to affect monocyte ingestion of zymosan particles or Er. Thus, the monocyte receptor for particulate activators that is specifically inhibited by beta-glucan at a rate compatible with a phagocytic process and that recognizes beta-glucans but not alpha-glucans, mannan, or galactan is a beta-glucan receptor.     

Effect of fibronectin fragments on macrophage phagocytosis of gelatinized particles

Rat plasma fibronectin enhances the binding and ingestion of gelatin-coated, formalin-fixed, or tanned sheep erythrocytes by elicited rat peritoneal macrophages. Fibronectin binding to the gelatinized erythrocytes is required for this enhancement, because macrophages preferentially recognize the surface bound molecule. This enhancement of particle uptake by fibronectin required the presence of a renewable, trypsin-sensitive component(s) on the macrophage surface (fibronectin receptor). When subjected to plasminolysis for 3 hr, fibronectin was degraded into gelatin-binding fragments (170 to 210 kd) and a 25-kd nongelatin binding fragment. The 170 to 210 kd gelatin binding fragments retained uptake-enhancing activity but were less active on a weight and molar basis than intact, dimeric fibronectin. The nongelatin binding 25 kd fragment alone did not enhance uptake. These results indicate that the sites for interaction with both the gelatinized erythrocyte surface and macrophages are retained on 170 to 210 kd fragments. However, the fibronectin dimeric structure is required for maximal expression of opsonic activity.     

Monocyte-derived macrophage and alveolar macrophage fibronectin production and cathepsin D activity

Alveolar macrophages are thought to play an important role in ongoing tissue breakdown and repair processes in the normal lung. The secretion and regulation of cathepsin D (important for the final breakdown of collagen) and fibronectin (involved in the healing process) in human peripheral blood monocytes (PBM) and pulmonary alveolar macrophages (PAM) were investigated. Cathepsin D enzyme activity was measured by quantitating the TCA-soluble fragments of [3H]hemoglobin. Freshly isolated PBM contained less cell-associated cathepsin D activity than did freshly isolated PAM (314 +/- 35 micrograms/10(6) cells vs 381 +/- 35 micrograms/10(6) cells, respectively). After 7-10 days in culture, cell-associated enzyme levels in both PBM and PAM were significantly increased (P less than 0.001 for PBM; P less than 0.0001 for PAM). In addition, freshly isolated PAM secreted more cathepsin D than did freshly isolated PBM (5.8 +/- 3.2 micrograms/10(6) cells vs 0.83 +/- 0.83 micrograms/10(6) cells, P less than 0.02). In the presence of LPS (10 micrograms/ml), cell-associated cathepsin D was inhibited in both PBM and PAM. With the addition of gamma-IFN (500 U/ml), both cell-associated and secreted enzyme were increased in freshly isolated and 10-day-cultured PBM and PAM. In parallel studies, fibronectin secretion (by ELISA assay) in both PBM and PAM increased over time in culture. LPS had no effect on PBM or PAM secretion of human fibronectin while gamma-IFN increased PBM and PAM fibronectin levels. Thus, both macrophage cathepsin D activity and fibronectin secretion are increased by gamma-interferon while macrophage cathepsin D activity, but not fibronectin secretion, is decreased by LPS. These studies demonstrate that human macrophage cathepsin D activity is actively modulated by inflammatory mediators and that macrophage mediators of tissue breakdown and repair are not modulated synchronously.     

Mapping the binding of monoclonal antibodies to histone H5

The binding sites of nine monoclonal antibodies along the polypeptide chain of histone H5 were mapped. Immunoblotting experiments with peptides generated from H5 by trypsin digestion, N-bromosuccinimide cleavage, and cyanogen bromide cleavage revealed that all of the monoclonal antibodies reacted with the globular region of H5 which is encompassed by amino acid residues 22-98. Within this globular segment, the epitopes could be subdivided into three regions. Monoclonals 1G11, 2E5, and 2H5 bind to residues 28-31. The close proximity of the epitopes was verified by a competitive enzyme-linked immunosorbent assay and by their binding pattern to a tryptic digest of H5. Monoclonals 4C6, 6E12, and 2E12 bind to a region encompassed by amino acids 28-53 while monoclonals 4H7, 1C3, and 3H9 bind to a region encompassed by residues 53-98. Precise localization of the epitopes in the primary sequence of H5 will allow detailed studies on the mode of binding of H5 to core particles in chromatin.     

Evidence for preferential proteolytic cleavage of one of the two fibronectin subunits and for immunological localization of a site distinguishing them

Purified plasma fibronectin was digested sequentially by thrombin and cathepsin G or by cathepsin G alone and the degradation products and their gelatin-binding and heparin-binding fractions were analyzed in NaDodSO4-polyacrylamide gel electrophoresis followed by immunoblotting with a defined monoclonal anti-fibronectin antibody. In early cathepsin G digests, several gelatin-binding fragments were detected: a few large (Mr greater than or equal to 150 000) polypeptides and fragments of Mr = 85 000, 72 000, 64 000 and 40 000. The 85 000-Mr and 64 000-Mr fragments appeared as closely spaced doublets and reacted with the antibody while the 72 000-Mr and 40 000-Mr fragments did not. Therefore the 64 000-Mr fragments are likely to be derived from the 85 000-Mr fragments. Three large fragments that bound to heparin, but not to gelatin were detected: Mr = 145 000, 135 000 and 120 000. Of these only the 135 000-Mr peptide reacted with the antibody. When fibronectin was digested with thrombin, polypeptides of Mr = 180 000-200 000 and a 30 000-Mr NH2-terminal fragment were produced. Cathepsin G added to this mixture further cleaved the fragments to a digestion pattern resembling that obtained from intact fibronectin except that the 85 000-Mr and 64 000-Mr fragments appeared as single bands and the amount of the 72 000-Mr fragment was reduced. The results suggest that thrombin cleaves the 30 000-Mr fragment preferentially from the NH2-terminal end of one of the two subunits of fibronectin and that the 85 000-Mr, 72 000-Mr and 64 000-Mr fragments obtained by the additional cathepsin G digestion were derived from the other chain. The results are consistent with the model that the antigenic determinant resides 72 000-85 000 Da from the NH2-terminus and is cleaved by cathepsin G alternatively at one of its sides. Thus, the components of the 85 000-Mr and 64 000-Mr doublets are derived from different subunits and the region located by the antibody may be responsible for the difference in their migration in the polyacrylamide gel.     

Augmentation of phagocytosis by a specific fibronectin fragment that links particulate activators to the fibronectin adherence receptor of human monocytes

Intact human plasma fibronectin of 44,000 m.w. and a fibronectin fragment of 180,000 m.w. promote dose-dependent adherence of gelatin-coated particles to human monocytes without phagocytosis. Both of these proteins, however, augment monocyte ingestion of gelatin-coated targets that are particulate activators of the alternative complement pathway or of nonactivators bearing IgG. Unlike intact fibronectin, the 180,000 m.w. fragment also binds directly to particulate activators that lack gelatin to augment their phagocytosis by human monocytes. Prior attachment to monocytes of gelatin-coated sheep erythrocytes bearing increasing concentrations of intact fibronectin decreases in a dose-dependent fashion the capacity of these monocytes to engage in augmented phagocytosis of particulate activators opsonized with the 180,000 m.w. fibronectin. Occupation of the monocyte fibronectin receptors with particle-bound, intact fibronectin does not decrease monocyte phagocytosis of plain particulate activators or of IgG-coated particles. Thus, the 180,000 m.w. fibronectin fragment both directly opsonizes particulate activators and interacts with monocyte fibronectin receptors to promote particle adherence, thereby enhancing phagocytosis through a concerted action with the distinct receptors for particulate activators.     

Structural requirement for the recognition of luteinizing hormone releasing hormone (LHRH) by monoclonal and conventional anti-LHRH antibodies

Immunochemical studies on monoclonal and conventional anti-LHRH antibodies have been reported. The association constants (Ka) were in the order of 10(9)-10(10) L/mol when calculated from Scatchard and Steward-Petty plots. Heterogeneity indices (a) calculated from Sips plot were nearly 1.0, indicating the homogeneous nature of monoclonals. Most of the conventional anti-LHRH produced by monkey, baboon, rabbit, and dog, by immunization using LHRH linked to tetanus toxoid by the carbodiimide condensation method, showed a single slope in Scatchard analysis (except two dog antisera) and a values were nearly 1.0. Monoclonals and conventionals reacted most strongly with native LHRH(NH2). Monoclonals showed poor reactivity with LHRH free acid and LHRH fragments containing free acid. The C-terminus tetrapeptide 7-10 showed 10 times more reactivity than tripeptide 4-6. The heptapeptide 4-10 showed 100 and 1000 times more reactivity than the tetra- and tri-peptide, respectively. Introduction of the tripeptide pGlu-His-Trp-OH to heptapeptide 4-10 caused five times more inhibition in reactivity than the heptapeptide. Conventional anti-LHRH antibodies manifested specificity to the C-terminus of LHRH. These sera did not react with LHRH free acid and LHRH fragments of sequence 4-6, 7-10, and 4-10. The complete loss of reactivity of conventional antibodies and poor reactivity of monoclonal antibodies was partly regained when LHRH free acid was coupled to Lys, Lys-MDP, or (Ala)2-tuftsin, suggesting that for monoclonals and conventionals the antigenic determinant was confined to the conformation involving the C-terminus amide of LHRH.     

Production and Characterization of Monoclonal Antibodies against Aspartate Aminotransferase-P(2) from Lupin Root Nodules

Twenty-one monoclonal antibodies were raised against the aspartate aminotransferase-P₂ isoenzyme from root nodules of Lupinus angustifolius [L.] cv Uniharvest. Induction of this isoenzyme is positively correlated with the onset of N₂ fixation in effective root nodules and is associated with the assimilation of ammonia by the plant in the Rhizobium-legume symbiosis. The monoclonal antibodies produced were all of the IgG class, recognized five different epitopes on the protein, and represented greater than 90% of the available epitopes. These epitopes were not unique to lupin nodule aspartate aminotransferase-P₂ but were shown to be present on the enzyme from tobacco leaves and potato. Four of the epitopes were conformational with a fifth epitope recognized by the appropriate monoclonals in both its native and denatured forms. None of the monoclonal antibodies produced reacted with Rhizobium Iupini NZP2257 extracts. Antibodies against two epitopes showed some cross-reaction with the constitutive aspartate aminotransferase-P₁ isoenzyme also found in lupin root nodules. However, affinity of these monoclonals for AAT-P₁ was three orders of magnitude lower than for AAT-P₂. Monoclonals against the other epitopes appeared to be specific for aspartate aminotransferase-P₂.     

Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment

Monocytes and lymphocytes form a second wave of infiltrating blood leukocytes in areas of tissue injury. The mechanisms for monocyte accumulation at these sites are not completely understood. Recently, however, fragments from extracellular matrix proteins including collagen, elastin, and fibronectin have been shown to induce monocyte chemotaxis. In this report we demonstrate that chemotactic activity for human monocytes is expressed when a 120-kDa fragment containing the RGDS cell-binding peptide is released from intact fibronectin or from larger fibronectin fragments. Monocytes, either from mononuclear cell Ficoll-Hypaque preparations (10-20% monocytes, 89-90% lymphocytes) or from elutriation preparations (95% monocytes, 5% lymphocytes), but not lymphocytes, migrated toward 120-kDa fragment preparations (10(-7) M) in blind-end chambers when the cells were separated from the chemoattractant by a 5-micron pore polycarbonate filter either alone or overlying a 0.45-micron pore nitrocellulose filter. Neutrophils migrated toward zymosan-activated serum but not toward 10(-5)-10(-8) M concentrations of the 120-kDa fragment. Intact fibronectin had no chemotactic activity for human monocytes. Fibronectin was isolated from citrated human plasma by sequential gelatin-Sepharose affinity and DEAE ion-exchange chromatography in the presence of buffers containing 1 mM phenylmethylsulfonyl fluoride to prevent fragmentation. Controlled enzymatic digestion with thermolysin cleaved fibronectin into 30 kDa fibrin, 45 kDa collagen, and 150/160-kDa cell and heparin domains. Upon prolonged digestion, purified 150/160-kDa fragments were cleaved into 120-kDa cell and 30/40-kDa heparin-binding fragments. Even though the intact fibronectin molecule, the 150/160-kDa fragments, and the 120-kDa fragment, have cell binding activity for Chinese hamster ovary fibroblasts, only the 120-kDa fragment expressed chemotactic activity for human monocytes. Thus, the 120-kDa fibroblastic cell-binding fragment contains a cryptic site for monocyte chemotaxis which is expressed upon enzymatic cleavage of fibronectin.     

Transglutaminase-sensitive glutamine residues of human plasma fibronectin revealed by studying its proteolytic fragments

The sites of transglutamination of fibronectin and fibronectin fragments, by coagulation factor XIIIa and tissue transglutaminase, were studied. It was shown that the intact fibronectin molecule has two sites sensitive to coagulation factor XIIIa and four sites sensitive to tissue transglutaminase: 180--190-kDa gelatin/heparin-binding fragments, 2 and 5--6 sites; 29-kDa heparin-I/fibrin-I-binding N-terminal fragments, 1 and 2 sites; 70-kDa gelatin-binding fragments, 0 and 1 site; 60-kDa cell-binding central fragments, 1 and 3--4 sites; 60-kDa, 45-kDa, 30-kDa heparin-II-binding C-terminal fragments, 1 and 2 sites. Thus, we have found a new coagulation-factor-XIIIa-sensitive site localized in the cell-binding central fragment, inaccessible to enzyme in the intact fibronectin molecule. Tissue transglutaminase appeared to interact with all of the three coagulation-factor-XIIIa-sensitive sites and, in addition, some others which are either available on the intact molecule or can be revealed only in proteolytic fragments of the fibronectin. We suggest that interdomain and intersubunit interactions in the intact fibronectin molecule account for the masking of glutamine residues potentially accessible to transglutaminases.     

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Fibronectin's cell-adhesive domain and an amino-terminal matrix assembly domain participate in its assembly into fibroblast pericellular matrix

Fibroblasts organize the modular cell-adhesive glycoprotein fibronectin into a highly structured pericellular matrix by poorly understood mechanisms. Previous studies implicated an amino-terminal domain in matrix assembly and suggested that fibronectin's cell-adhesive domain and the corresponding fibroblast receptor were not involved in this process. To further elucidate the fibronectin region(s) involved in matrix assembly, we mapped a library of proteolytic fragments and antibodies to various fibronectin domains. The fragments and antibodies were used to probe the role of fibronectin's amino-terminal and cell-adhesive domains in a fibroblast matrix assembly assay. We found that fibronectin fragments including the first 25-kDa sequence of fibronectin and antibodies to amino-terminal domains inhibited pericellular matrix assembly. Polyclonal antibodies to the 40-kDa collagen binding domain following the 25-kDa amino-terminal domain also inhibited matrix assembly. However, collagen binding is not required for matrix assembly as neither monoclonals blocking collagen binding nor purified collagen binding domains themselves inhibited matrix assembly. Therefore, the amino-terminal region of fibronectin contains a site important in matrix assembly, and most activity is present in the first 25-kDa of fibronectin. Fibronectin's cell-adhesive domain and the fibroblast receptor binding to this domain also play an important role in fibronectin matrix assembly. Apart from a monoclonal antibody to the amino-terminal domain, only monoclonal antibodies binding to fibronectin's cell-adhesive domain and inhibiting cell adhesion also inhibited matrix assembly. In addition a 105-kDa fragment containing the cell-adhesive domain inhibited matrix assembly. We conclude that at least two discrete and widely separated sites in fibronectin with different binding properties--the carboxyl-terminal fibroblast cell-adhesive domain and an amino-terminal matrix assembly domain localized primarily within the first 25 kDa--are required for fibronectin pericellular matrix assembly by fibroblasts. Fibronectin's cell-adhesive domain and its cell surface-receptor complex appear to be involved in the matrix assembly process prior to a step involving the amino-terminal domain. We believe that this step is likely to be the initiation of cell-associated fibronectin fibril formation by the fibronectin-adhesive-receptor complex.     

Enzymatic modifications of human plasma fibronectin in relation to opsonizing activity

Plasma fibronectin is one of the largest plasma proteins (Mr approximately 440 000), comprising two approximately equal polypeptide chains which are held together by a disulfide linkage near the C-terminal end of the molecule. The binding of gelatinized latex beads to liver slices as well as the internalization of these particles by macrophages, in the presence of heparin, is greatly enhanced by fibronectin. The question as to whether the entire covalent structure of fibronectin was necessary for opsonizing activity was approached by limited proteolytic degradations of the molecule. Patterns of controlled digestion with trypsin, cathepsin D, Staphylococcus aureus protease, and plasmin all indicate that the minimal unit necessary for retention of opsonic activity is some large (Mr 200 000 and 190 000) single-chain entity. Treatment with plasmin proved to be the most reliable procedure for generating the active split product which could be readily separated from the inactive, disulfide-containing C-terminal fragment. Incorporation of dansylcadaverine into plasma fibronectin (3.5 mol/mol of protein) by fibronoligase (coagulation factor XIIIa) did not affect the opsonic activity of the protein.     

The biochemical and functional heterogeneity of circulating human plasma fibronectin

Human plasma fibronectin purified by affinity chromatography, consisted of homogeneous 215 kD protein subunits when assessed by SDS polyacrylamide gel electrophoresis. On isoelectric focusing however, 5 separate fractions were present, with isoelectric points ranging from 5.6 to 6.1. Isoelectric focusing and immunofixation of native plasma produced similar but not identical appearances. Only 15% of the total plasma fibronectin opsonically stimulated the ingestion of Saccharomyces cerevisiae by human peripheral blood monocytes, and this opsonic fibronectin was confined to the fraction with an isoelectric point of 6.1.     

Monoclonal antibody characterization of two distant sites required for function of the central cell-binding domain of fibronectin in cell adhesion, cell migration, and matrix assembly

Site-directed mutagenesis studies have suggested that additional peptide information in the central cell-binding domain of fibronectin besides the minimal Arg-Gly-Asp (RGD) sequence is required for its full adhesive activity. The nature of this second, synergistic site was analyzed further by protein chemical and immunological approaches using biological assays for adhesion, migration, and matrix assembly. Fragments derived from the cell-binding domain were coupled covalently to plates, and their specific molar activities in mediating BHK cell spreading were compared with that of intact fibronectin. A 37-kD fragment purified from chymotryptic digests of human plasma fibronectin had essentially the same specific molar activity as intact fibronectin. In contrast, other fragments such as an 11.5-kD fragment lacking NH2-terminal sequences of the 37-kD fragment had only poor spreading activity on a molar basis. Furthermore, in competitive inhibition assays of fibronectin-mediated cell spreading, the 37-kD fragment was approximately 325-fold more active than the GRGDS synthetic peptide on a molar basis. mAbs were produced using the 37-kD protein as an immunogen and their epitopes were characterized. Two separate mAbs, one binding close to the RGD site and the other to a site approximately 15 kD distant from the RGD site, individually inhibited BHK cell spreading on fibronectin by greater than 90%. In contrast, an antibody that bound between these two sites had minimal inhibitory activity. The antibodies found to be inhibitory in cell spreading assays for BHK cells also inhibited both fibronectin-mediated cell spreading and migration of human HT-1080 cells, functions which were also dependent on function of the alpha 5 beta 1 integrin (fibronectin receptor). Assembly of endogenously synthesized fibronectin into an extracellular matrix was not significantly inhibited by most of the anti-37-kD mAbs, but was strongly inhibited only by the antibodies binding close to the RGD site or the putative synergy site. These results indicate that a second site distant from the RGD site on fibronectin is crucial for its full biological activity in diverse functions dependent on the alpha 5 beta 1 fibronectin receptor. This site is mapped by mAbs closer to the RGD site than previously expected.     

Localization and chemical synthesis of fibronectin peptides with melanoma adhesion and heparin binding activities

Tumor cell adhesion to the extracellular matrix is an important consideration in tumor metastasis. Recent results show that multiple adhesion-promoting domains for melanoma cells can be purified from proteolytic digests of fibronectin [McCarthy, J. B., Hagen, S. T., & Furcht, L. T. (1986) J. Cell Biol. 102, 179-188]. Monoclonal antibodies were generated against a tryptic/catheptic 33K heparin binding fragment of fibronectin derived from the carboxyl terminal of the A chain. This region contains a tumor cell adhesion-promoting domain(s). The amino-terminal sequence was determined for this fragment, as well as a tryptic 31K fragment which is located to the carboxyl-terminal side of the 33K heparin binding fragment in A chains of fibronectin. The partial sequence data demonstrate that arginyl-glycyl-aspartyl-serine (RGDS) or the related arginyl-glutamyl-aspartyl-valine (REDV) is not present in the 33K heparin binding fragment, confirming earlier results which demonstrated that cells adhere to this fragment by an RGDS-independent mechanism. Two monoclonal antibodies, termed AHB-1 and AHB-2, recognized epitopes common to heparin binding fragments derived from the carboxyl terminus of both the A and B chains of fibronectin. Monoclonal antibody AHB-2 inhibited melanoma adhesion to the 33K heparin binding fragment of fibronectin in a concentration-dependent manner, whereas monoclonal antibody AHB-1 had no effect on adhesion to this fragment. Neither monoclonal antibody inhibited adhesion to intact fibronectin. However, monoclonal AHB-2 potentiated the inhibitory effect of suboptimal levels of exogenous RGDS on cell adhesion to intact fibronectin. AHB-2 recognized an epitope common to both the A- and B-chain carboxyl-terminal heparin binding region of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Different effects of fibronectin on the phagocytosis of Staphylococcus aureus and coagulase-negative staphylococci by murine peritoneal macrophages

Fibronectin is known to be an important factor in colonization by Staphylococcus aureus of host tissues as well as other extracellular matrix proteins such as collagen and laminin. We investigated the effect of fibronectin on the phagocytosis of the S. aureus Cowan I strain by macrophages and of coagulase-negative staphylococci (CNS) strains for comparison. Fibronectin-reduced serum in place of normal serum lowered the phagocytic activity of the macrophages on the Cowan I strain. Purified fibronectin enhanced the phagocytic activity of the strain in a dose-dependent manner. On the other hand, fibronectin did not show any opsonic effect on the ingestion of CNS strains, though the binding of fibronectin occurred equally well in CNS strains and the Cowan I strain. Fibronectin-binding protein (FnBP), the specific fibronectin receptor on the surface on S. aureus, was detected in both the Cowan I strain and CNS strains. Polymerase chain reaction confirmed that not only the Cowan I strain, but also CNS strains possessed the FnBP gene. These results indicate that fibronectin shows an opsonic effect on the S. aureus, Cowan I strain but not on CNS strains, and suggest that the binding of fibronectin to FnBP is not sufficient for efficient phagocytosis of the staphylococci strains by macrophages.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Characterization of a fragment of bovine von Willebrand factor that binds to platelets

Bovine von Willebrand factor was digested with human plasmin in order to isolate and characterize a fragment that can bind to human platelets. A terminal plasmin digest of bovine von Willebrand factor is composed of five fragments, ranging in relative molecular weight (Mr) from 250,000 to 35,000. The major fragment has a Mr of 250,000 and consists of four disulfide-linked polypeptide chains with Mr from 69,000 to 35,000. The Mr 69,000 and 49,000 polypeptides possess carbohydrate moieties, as indicated by their reaction with periodate-Schiff reagent. Gel filtration studies suggest that, at physiological ionic strength, four of the Mr 250,000 fragments associate into a limited noncovalent oligomer. Monoclonal antibodies were prepared against native von Willebrand factor and used to characterize the distribution of epitopes on native vWF and the Mr 250,000 major fragment. Two of the monoclonal antibodies that recognize the major fragment (2 and H-9) inhibit platelet agglutination. The Mr 250,000 fragment binds to human platelets, and the binding is inhibited by monoclonal antibodies 2 and H-9. The Mr 250,000 fragment does not agglutinate platelets, consistent with a requirement for high molecular weight oligomers of von Willebrand factor for platelet agglutination. The Mr 250,000 fragment can compete with intact, bovine von Willebrand factor for binding to human platelets. However, its affinity is one-tenth that of intact von Willebrand factor.