Developmental regulation of a secreted gelatin-binding protein during myogenesis in vitro

Collagen has a stimulatory effect on the differentiation of skeletal muscle cells in culture. Putative collagen-binding proteins were isolated from detergent-solubilized cultures of the L6 rat muscle cell line and primary clonal cultures of human skeletal muscle satellite cells, using gelatin-Sepharose affinity chromatography. In addition to fibronectin, which has been reported by others to be synthesized by cultured muscle cells, we found that muscle cultures synthesized gelatin-binding proteins of lower apparent molecular weight. Only one of these proteins was secreted into the growth medium and bound to type I collagen. Binding of this protein to gelatin and collagen-Sepharose was resistant to repeated washing with 1 M NaCl and nonionic detergent. The secreted gelatin-binding protein had an apparent molecular weight of 63,000-72,000, depending upon the conditions of electrophoresis. The lack of reactivity of the secreted protein with polyclonal antisera against fibronectin, the lack of effect of protease inhibitors on its appearance in the medium, and the rapid de novo production of the protein during pulse labeling with radioactive methionine indicated that it was not a fibronectin fragment. The rate of synthesis of the secreted gelatin-binding protein increased markedly during the myogenesis of rat and human cultures.     

A novel lectin-independent interaction of P fimbriae of Escherichia coli with immobilized fibronectin

Binding of P fimbriae of uropathogenic Escherichia coli to purified human fibronectin and human placental type IV collagen was studied. In an enzyme immunoassay, purified P fimbriae bound strongly to immobilized intact fibronectin and to the aminoterminal 30-kDa fragment and the 120-140-kDa carboxyterminal fragments of fibronectin. Binding to the gelatin-binding 40-kDa fragment of fibronectin was considerably weaker. No binding to immobilized type IV collagen was seen. The interaction between P fimbriae and immobilized fibronectin was not inhibited by alpha-D-Gal-(1-4)-beta-D-Gal-1-O-Me, a receptor analog of P fimbriae. Moreover, a mutated P fimbria lacking the lectin activity behaved similarly in the adherence assays. Recombinant strains expressing the corresponding cloned fimbriae genes bound to immobilized fibronectin, but no binding to soluble 125I-labelled fibronectin was found. The results suggest that P fimbriae interact with immobilized fibronectin and that the binding mechanism does not involve the lectin activity of the fimbriae.     

Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments

We have identified the biological activity of three polypeptides released by limited proteolysis of human plasma fibronectin by leukocyte elastase. A Mr = 140,000 peptide contains cell-spreading activity; a Mr = 60,000 peptide mediates binding to denatured collagen (gelatin), and a Mr = 29,000 peptide contains glutaminyl residues responsible for the transglutaminase (blood coagulation factor XIIIa)-catalyzed incorporation of amines. More extensive proteolysis yielded numerous peptides, including a Mr = 40,000 peptide derived from the Mr = 60,000 peptide which retains gelatin-binding activity. Quantification of the gelatin-binding peptides is consistent with two binding sites per dimeric fibronectin molecule of Mr = 440,000. Both Mr = 60,000 and 40,000 gelatin-binding peptides were enriched with half-cystine residues, containing 28 and 25, respectively, but devoid of cysteine. This, coupled with the electrophoretic behavior of both peptides, was consistent with the presence of intramolecular disulfide bonds in the gelatin-binding domain. Intact fibronectin contains 1 free cysteine residue/monomer, as recently described. This cysteine reacts with 5,5'-dithiobis(2-nitrobenzoic acid) very slowly under nondenaturing conditions but rapidly when fibronectin is denatured. The free cysteine is located in the Mr = 140,000 peptide. While the Mr = 40,000 and 60,000 gelatin-binding peptides bind to gelatin with an affinity about 30-fold and 5-fold less than intact fibronectin (based on a monomeric fibronectin Mr = 220,000), neither gelatin-binding peptide supports spreading of fibronectin-deficient test cells on gelatin or tissue culture plastic substrates. The purified Mr = 140,000 peptide supported cell spreading on plastic, retaining about one-half of the spreading activity of intact fibronectin on a weight basis. These data confirm recent results, suggesting multiple, protease- resistant domains with discrete biological functions within fibronectin. Our results, together with established data, suggest a model for the location of the transglutaminase-reactive glutaminyl residues, gelatin binding, and cell-adhesive domains in fibronectin. The release of univalent, biologically active fibronectin fragments by elastase, a major physiologically released inflammatory protease of human leukocytes, suggests a new potential mechanism for alteration of cell connective tissue interactions at sites of inflammation in vivo.     

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.     

The collagen-binding site of type-II units of bovine seminal fluid protein PDC-109 and fibronectin

A single type-II domain has been isolated by limited proteolysis of the collagen-binding bovine seminal fluid protein, PDC-109. The 45-residue fragment corresponding to the second type-II domain of the parent molecule was found to have retained affinity for immobilized collagen, indicating that this minidomain carries critical regions of the collagen-binding site. Studies on various fragments of fibronectin have also implicated the two type-II units of this molecule in collagen-binding. In the present work we have found that type-II domains of human fibronectin, expressed in Escherichia coli as beta-galactosidase fusion proteins, bind specifically to immobilized collagen.     

Human placental (fetal) fibronectin: increased glycosylation and higher protease resistance than plasma fibronectin. Presence of polylactosamine glycopeptides and properties of a 44-kilodalton chymotryptic collagen-binding domain: difference from human plasma fibronectin

Human placental fibronectin was isolated from fresh term placenta by urea extraction and purified by gelatin affinity chromatography. A 44-kDa chymotryptic fragment, also purified by gelatin affinity chromatography, gave a broad, diffuse band on polyacrylamide gel electrophoresis, whereas the analogous 43-kDa fragment from human plasma fibronectin migrated as a defined, narrow band. Upon extended treatment with endo-beta-galactosidase from Escherichia freundii, the 44-kDa chymotryptic gelatin-binding fragment from placental fibronectin changed its behavior on gel electrophoresis and migrated as a narrower, more defined band. The carbohydrates on human placental fibronectin contained a large percentage of polylactosamine structures, part of which occurred on the gelatin-binding fragment, comprising almost twice as much carbohydrate as plasma fibronectin. NH2-terminal amino acid sequence analysis of the chymotryptic gelatin-binding fragments from both fibronectins showed the first 21 residues to be identical. Tryptic and chymotryptic peptide maps of the gelatin-binding fragment from placental fibronectin, however, showed differences including several protease-resistant domains not found in the analogous fragment from plasma fibronectin. Intact placental fibronectin contains 20,000 Da of carbohydrate, whereas plasma fibronectin contains 11,000 Da. Placental fibronectin is more protease-resistant than plasma fibronectin, possibly due to the additional carbohydrate. Polyclonal antibodies against either fibronectin completely cross-react with amniotic fluid fibronectin, placental fibronectin, and plasma fibronectin upon Ouchterlony immunodiffusion. Human fibronectins of putatively the same polypeptide structure are, therefore, glycosylated in a dramatically different fashion, depending on the tissue of expression. If the patterns of glycosylation comprise the only difference in the glycoprotein, this may confer the characteristic protease resistance found for each of the fibronectins.     

Collagen-binding domain of human plasma fibronectin contains a latent type-IV collagenase.

Cleavage of the 45-kDa gelatin-binding fragment of human plasma fibronectin with fibronectinase resulted in the activation of two forms of metalloproteinase with different substrate specificities. The 40-kDa FN-type-IV collagenase A degrades heat-denatured type-I collagen, laminin and also native collagen type IV. The 27-kDa FN-type-IV collagenase B degrades native collagen type IV, but it does not cleave laminin and only poorly degrades gelatin. Both enzymes begin with the same N-terminal sequence VYQPQPH- (residues 262-268 of fibronectin) but, contrary to the FN-type-IV collagenase A, the FN-type-IV collagenase B has lost the C-terminal region of type I repeats, where the major gelatin-binding determinants of fibronectin are located. The FN-type-IV collagenases A and B are sequentially similar to the middle domain (domain II) of collagenase type IV, secreted by H-ras-transformed human bronchial epithelial cells. Substrate and inhibition specificity of FN-type-IV collagenase A and B are different from those of FN-gelatinase and FN-laminase, isolated previously from the central and C-terminal fibronectin domains, respectively. The substrate specificity of both enzymes, characterized in this study, is also different from that of already known matrix-degrading metalloproteinases.     

The PDC-109 protein from bovine seminal plasma is similar to the gelatin-binding domain of bovine fibronectin and a kringle domain of human tissue-type plasminogen activator

PDC-109, a protein of unknown function, is a major component of bovine seminal plasma. Using a computer program designed to detect evolutionary relationships between proteins, I find that the PDC-109 protein is similar to the gelatin-binding domain of bovine fibronectin and part of a kringle domain of human tissue-type plasminogen activator (t-PA). The computer-based comparison of the amino acid sequence of PDC-109 with that of the gelatin-binding domain of fibronectin and part of the second kringle domain of t-PA yields scores that are 15.5 standard deviations and 7.8 standard deviations higher, respectively, than were obtained with a comparison of randomized sequences of these proteins. The probability (p) of getting these scores by chance is less than 10(-50) and 3 X 10(-15), respectively. The similarity between the amino acid sequences of PDC-109 and the gelatin-binding domain in fibronectin and the kringle of t-PA suggests some approaches for identifying the functions of PDC-109. Both t-PA and the gelatin-binding domain of fibronectin have adhesive functions, and the gelatin-binding domain promotes viral transformation of fibroblasts in culture. These functions may be associated with the PDC-109 protein.     

Role of the I-9 and III-1 modules of fibronectin in formation of an extracellular fibronectin matrix.

Cultured fibroblasts bind soluble protomeric fibronectin and mediate its conversion to insoluble disulfide-bonded multimers. The disulfide-bonded multimers are deposited in fibrillar pericellular matrix. Antifibronectin monoclonal antibodies were analyzed to identify domains of fibronectin required for assembly into matrix. Two antibodies, L8 and 9D2, inhibited binding and insolubilization of 125I-labeled plasma fibronectin by fibroblasts but did not inhibit binding of labeled amino-terminal 70-kDa fragment of fibronectin to matrix assembly sites. Immunoblotting of fibronectin fragments showed that the epitope for 9D2 is in the first type III homology sequence (III-1) whereas the epitope for L8 requires that the last type I sequence of the gelatin binding region (I-9) be contiguous to III-1 and is sensitive to reduction of disulfides in I-9. A 56-kDa gelatin-binding thermolysin fragment of fibronectin that contains III-1 and the L8 and 9D2 epitopes inhibited binding of fibronectin to cell layers 10-fold better than a 40-kDa gelatin-binding fragment that lacks III-1 and the antigenic sites. This 56-kDa fragment, however, did not bind specifically to cell layers. These results indicate that the I-9 and III-1 modules of fibronectin form a functional unit that mediates an interaction, perhaps between protomers, important in the assembly of fibronectin.     

Immunological cross-reactivity between human tripeptidyl peptidase II and fibronectin.

Tripeptidyl peptidase II (TPP II) is a large intracellular exopeptidase with an active site of the subtilisin type. Affinity-purified hen antibodies against human erythrocyte TPP II cross-reacted with fibronectin in an immunoblot analysis. Furthermore, antibodies against human fibronectin cross-reacted with TPP II. Antibodies against a 65 kDa cell-binding fragment of fibronectin specifically reacted with TPP II, whereas antibodies against the collagen-binding domain, the main heparin-binding domain or the N-terminal fibrin-binding domain did not react. Moreover, the affinity-purified antibodies against TPP II reacted with a 105 kDa cell-binding fragment of fibronectin but not with the fibrin-binding domain or the collagen-binding domain. When native TPP II was dissociated into smaller units through dialysis against a dilute Tris buffer, it could be digested by chymotrypsin into three stable fragments of 70 kDa, 42 kDa and 20 kDa. It could be demonstrated that the 42 kDa fragment was specifically recognized by antibodies against the 65 kDa cell-binding fragment of fibronectin. Furthermore, labelling with di-[3H]isopropyl phosphorofluoridate and N-terminal sequence determination showed that the 70 kDa fragment contained the active-site serine residue. In conclusion, our findings suggest that one domain of the TPP II molecule bears structural resemblance to a cell-binding fragment of fibronectin.     

Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.     

Production of a biologically active epidermal growth factor fusion protein with high collagen affinity

Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner. In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain. A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system. This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl. In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin. FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges. These results suggested that our fusion protein with high collagen affinity would be useful for wound healing.     

A cell surface receptor complex for collagen type I recognizes the Arg- Gly-Asp sequence

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.     

Primary structure of a DNA- and heparin-binding domain (Domain III) in human plasma fibronectin

The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.     

Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules

Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Studies of extracellular fibronectin matrix formation with fluoresceinated fibronectin and fibronectin fragments

Fluorescein isothiocyanate conjugated human plasma fibronectin, 70-kDa collagen-binding, 60-kDa central, 60-kDa heparin-binding, 180-kDa heparin, collagen-binding fibronectin fragments and gelatin were used to study extracellular fibronectin matrix formation. Exogenous fibronectin, gelatin, 70-kDa collagen-binding and 180-kDa heparin, collagen-binding fragments were shown to be able to bind specifically to preexisting extracellular matrix of living fibroblasts. The results suggest that: (i) Fibronectin matrix formation may occur through a self-assembly process; (ii) the NH2-terminal part of fibronectin is responsible for fibronectin-fibronectin interaction during fibronectin fibril formation; (iii) plasma fibronectin may be the source for tissue fibronectin.     

Expression of a fusion protein containing human epidermal growth factor and the collagen-binding domain of <Emphasis Type="Italic">Vibrio mimicus</Emphasis> metalloprotease

Human epidermal growth factor (hEGF) is a polypeptide of 53 amino acids, is an important autocrine/paracrine factor in the human body, and is used in the pharmaceutical and cosmetics industries. We constructed a fusion hEGF protein with a collagen-binding domain (CBD) composed of 33 amino acids from Vibrio mimicus metalloprotease (VMCBD). The CBD segment of the metalloprotease was fused at the C terminus of the hEGF protein. The recombinant fusion protein was expressed in Escherichia coli and purified. The purified hEGF protein promoted greater growth of human/A-431 cells than did the control hEGF. The fusion EGF protein also showed collagen-binding activity with type I collagen. In contrast, hEGF did not bind to type I collagen. These results suggest that recombinant hEGF protein fused to VMCBD may be able to remain for a long period at injured epidermal tissue acting as a healing agent.     

Highly purified, functionally active human fibronectin preparation

Fibronectin has been purified by gelatin-Sepharose affinity chromatography from fresh frozen human plasma. The bound fibronectin was eluted with 3 M urea. The purity of the fibronectin obtained has been checked on (immunoelectrophoresis, polyacrylamide gel electrophoresis, FPLC). Biological activity of the purified molecule has been monitored by means of three assays: quantitation of the gelatin-binding activity by ELISA, quantitation of the fibronectin-mediated attachment of fibroblasts on plastic and evaluation of the opsonic activity (uptake of gelatin latex particles by a murine macrophage line). When deep-frozen, fibronectin retains all of its properties. This highly purified and functional fibronectin fulfills the basic requirements for a standard reagent. It will allow to investigate physicochemical and functional alterations of various fibronectins.     

Identification and characterization of collagen-binding activity in Streptococcus mutans wall-associated protein: a possible implication in dental root caries and endocarditis

Streptococcus mutans is implicated in coronal and dental root decay, and in endocarditis. Comparative study of the amino acid sequence of S. mutans 47 kDa wall-associated protein A (WapA) revealed a collagen-binding domain (CBD) at the N-terminal region. Recombinant AgA (WapA truncated at the carboxyterminal end) was isolated, biotin-labeled, and analyzed by Solid Phase Binding Assay. The results showed that biotin-labeled AgA bound significantly and in a dose-dependent manner to immobilized collagen type I, and to a lesser extent to fibronectin, but not to collagen type IV or laminin. Binding of biotin-labeled S. mutans cells to collagen-coated surfaces was significantly inhibited by antibody to WapA or AgA (P<0.001). The results obtained confirmed the collagen-binding activity of CBD in AgA and WapA, and suggested that WapA may be used, not only as a vaccine against coronal and dental root caries, but also against S. mutans-mediated endocarditis.