Approximately one-half of the amino acid sequence (911 amino acid residues out of 1,880 expected) for bovine plasma fibronectin (cold-insoluble globulin) has been determined. Three types of internal homology were identified, showing that a number of partial gene duplications (multiplications) have occurred during the evolution of this protein. Digestion of fibronectin with plasmin results in major fragments with molecular masses of 29, 170,23, and 6 kilodaltons (kDal). The NH2-terminal 29-kDal fragment consists of259 residues ordered as five mutually homologous domains (type I homology) with two disulfide bonds in each domain. The 170-kDal fragment shows two to three bands after NaDodSO4 gel electrophoresis, indicating heterogeneity. This fragment contains the gelatin binding site and the strong heparin binding site present in fibronectin. Digestion of the 170-kDal fragment with chymotrypsin liberates a 45-kDal fragment that also binds to gelatin. This fragment contains at least one domain oftype I homology and two domains of type II homology. Further digestion of the 170-kDal fragment with chymotrypsin results in the formation of a 30-kDal fragment that retains the heparin binding activity. This fragment contains sequences constituting type m homology. The 23-kDal fragment consists of 178 residues having three domains of type I homology. The 6-kDal fragment consists oftwo identical peptides of26 residues, and these two peptides are linked to each other by two disulfide bonds that form the interchain bridges. Another one ofthe peptides for which the sequence was determined links the COOH-terminus of the 29-kDal fragment to the NH2-terminus of the 170-kDal fragment. This and the fact that the COOH-terminal residue of the 6-kDal fragment is a glutamic acid residue order the four plasmin-digestion fragments as 29-, 170-, 23-, and 6-kDal in the intact fibronectin molecule.Fibronectin is a protein or a group of similar proteins found in blood plasma (1) and on the surface of different cell types (2) (for review, see refs. 3-5). The functions attributed to fibronectin, such as cell adhesion to substratum, cell spreading, opsonization of bacteria and other particulate matter, and wound healing, are all related to its affinity to cell surfaces. Binding to vastly different biological compounds, such as gelatin (6), glycosaminoglycans (7), DNA (8), fibrin (9), actin (10), sphingolipids (11), acetyl cholinesterase (12), amyloid P component (13), and complement factor Clq (14), has been demonstrated. Some of these binding sites have been found to be contained in specific fragments generated by limited proteolysis of fibronectin (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). Recently, a transformation-enhancing activity has been shown in the gelatin binding fragments offibronectin (25).Fibronectin is a substrate for blood coagulation factor XIIIa (26) and can be covalently crosslinked to fibrin (26), collagen (21), and the surface ofStaphylococcus aureus (22) by this transglutaminase. Fibronectin used to be known as cold...

The primary structure of the 2265 residues of bovine plasma fibronectin has been completed. The new sequences reported in this paper are residues 600-868 (269 residues), 1138-1217 (80 residues), 1518-1599 (82 residues) and 1868-2061 (194 residues). These sequences constitute six type I11 homology units and two nonhomologous connecting strands. Thus, there are fifteen type I11 homology units in plasma fibronectin. Evidence for two of the three splice variants found in rat liver cells [Schwarzbauer et al. (1983) Cell 35, 421 -4311 was obtained. No indication of the 'extra' type I11 domain present in some human fibroblast fibronectins [Kornblihtt et al. (1984) EMBO J. 3, 221 -2261 was found. Three carbohydrate groups (two glucosamine-based and one galactosamine-based) were identified, giving a total of eight carbohydrate groups in the longest splice variant of bovine plasma fibronectin. A second free sulfhydryl group (cysteine) was identified. This cysteine, like the first, is in a type I11 homology unit. HPLC patterns of peptides obtained from the C-terminal6-kDa fragment suggested that the interchain bridge pattern of fibronectin is antiparallel. The bovine plasma fibronectin sequence is highly homologous to the human cellular fibronectin sequence deduced from the cDNA sequence [Kornblihtt et al. (1985) EMBO J. 4,1755-17591. Fibronectin is a high-molecular-mass, multidomain glycoprotein found in a soluble form in blood plasma and in an insoluble form in loose connective tissue and basement membranes (reviewed in [l -31). Plasma fibronectin, which is synthesized and secreted by hepatocytes [4], and fibronectin synthesized by cultured fibroblasts are similar but not identical. Differences include carbohydrate content [5], fragmentation pattern [6], antigenicity [7] and number of chains [8, 91. Recently the existence of two fibronectin mRNA species from a human cell line was reported [lo]. One of these coded for an extra domain (ED) of 90 amino acid residues relative to the other mRNA. This ED insert seems to be absent in plasma fibronectin [lo] and may, therefore, account for at least some of the difference between cellular and plasma fibronectin.Plasma fibronectin consists of two subunits, which are linked to each other by two disulfide bonds near the C terminus [ll]. The subunits have a molecular mass of about 225 -250 kDa, that is, they vary in mass by 10 -25 kDa when subjected to SDS/polyacrylamide gel electrophoresis, under reducing conditions. The subunits have identical N-terminal and C-terminal sequences, showing that the difference is not due to limited proteolysis.Six cDNA clones, three from rat liver cells [12] and three from human cell lines [13-151, have been isolated corresponding to five different mRNAs. These differ in another area called IIICS (for type 111 connecting segment) C-terminal to the ED, and this area corresponds to the region in which (EC 3.4.21.19). differences between the two chains of fibronectin have been found (at the protein level) to be located [16-181. Recently the th...

Abstract. Thrombospondin (TSP) contains the ArgGly-Asp (RGD) sequence that is thought to be important for cell adhesion mediated by several cell-surface integrin receptors. The RGD sequence is located in the type 3 repeat region of TSP that has multiple Ca 2+ binding sites and is subject to a complex intramolecular thiol-disulfide isomerization. TSP that we isolated from thrombin-activated human platelets using buffers containing 0.1 mM Ca 2 § in which Cys TM is the major labeled cysteine, did not have RGD-inhibitable adhesive activity. However, one of our preparations of TSP and TSP purified following alternative procedures using >10.3 mM Ca 2 § did have RGD-inhibitable adhesive activity. Reduction of TSP with DTT, either before or after adsorption to surfaces, enhanced its adhesive activity. Reduced TSP supported robust cell spreading when coated at concentrations as low as 1 #g/ml, whereas "adhesive" TSP not treated with DTT was active at coating concentration of > 20 #g/ml and supported only modest cell spreading. Lower DTT concentrations were required for enhancement of the adhesive activity of TSP if Ca 2+ was chelated with EDTA. Cellular adhesion to DTT-treated TSP was inhibited by RGD-containing peptide and by mAb to a functional site of the av/$3 integrin. Cell blots of reduced proteolytic fragments of TSP localized the adhesive activity to the RGD-containing type 3 repeat region. These resuits suggest a novel mechanism for regulation of integrin-ligand interactions in which the ligand can isomerize between inactive and active forms.T hE originally described form of thrombospondin (TSP) ~ is a major glycoprotein of platelet a-granules released upon platelet activation and a major secreted product of many cultured cells (Mosher, 1990). According to Dr. Vishva Dixit (personal communication), platelet TSP is exclusively TSP1 when analyzed by isoform-specific antibodies. However, we call TSP purified from platelets just "TSP: Recently, another closely related but clearly distinct TSE termed TSP2, was identified from mouse and chicken cDNA libraries (Bomstein et al., 1991a,b;Lawler et al., 1991). Both TSP and TSP2 contain the cell adhesion sequence Arg-Gly-Asp (RGD) (Lawler and Hynes, 1986;Lawler et al., 1991). A number of RGD-containing extracellular proteins, such as vitronectin, fibronectin, and fibrinogen, unambiguously promote cell adhesion when coated onto substrata in a process mediated by cell surface integrin receptors (Ruoslahti and Pierschbacher, 1987). The adhesive activity of TSP, however, is more complicated. Different groups have tested TSP purified from platelet releasate for adhesive activity on various cells with contradictory and controversial results.Opposite results have been observed for the same cell type.1. Abbreviations used in this paper; BAE, bovine aorta endothelial; HBS, Hepes buffered saline; RGD, Arg-Gly-Asp; TSP, thrombospondin purified from platelets. Lahav (1988a) reported that TSP inhibits endothelial cell adhesion, while others found that TSP promotes endothelial cell adhe...

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Abstract. An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin . The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces . Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa) . Globulin fractions from normal plasma immunodepleted of high molecu-C ELL attachment and spreading on extracellular matrices are central events in a variety of biological phenomena such as embryogenesis and organogenesis, tumor metastasis, wound healing, and thrombus formation . These events are recapitulated by in vitro assays in which cells attach and spread on protein-coated surfaces. Such assays have given considerable insight about the molecular basis of cell attachment and spreading and led to the identification of the integrin group of heterodimeric adhesion receptors (Hynes, 1987;Ruoslahti and Pierschbacher, 1987;Hemler, 1990). The integrins interact with specific substrateadsorbed adhesion proteins, often by an Arg-Gly-Asp (RGD) sequence in the adhesion molecules .Increasing attention is being paid to the molecular basis of anti-adhesion . Regulatory substances such as transforming growth factor-ß and interleukin 8 influence adhesion by changing the expression of cell adhesion receptors by the adhering cell (Gimbrone et al., 1989;Heino and Massagu6, 1989) . Matrix-associated molecules such as tenascin (Chiquet-Ehrismann et al., 1988;Lotz et al., 1989;Spring et al., 1989), thrombospondin (Lahav, 1988;Hö6k, 1989), and SPARC (Sage et al ., 1989) inhibit adhesion when adsorbed to a potentially adhesive substrate . We were interested in whether anti-adhesive molecules other than thrombospondin are generated as a result of blood coagulation. Herein we report the purification and characterization ofapotent anti-adhesive globulin fromdextran sulfatetreated human plasma . This globulin inhibits adhesion of a variety of cells to vitronectin-or fibrinogen-coated substrates . Amino acid sequence analysis revealed that the glob-0 The Rockefeller University Press,

Fucosylated glycoproteins carrying ␣1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known ␣1-4-fucosyltransferase (FucT), and individuals who are deficient in this enzyme type as Lewis-negative on erythrocytes. We examined the mutational spectrum of the Lewis gene in Denmark and found 6 different mutations. Five, T59G, T202C, C314T, G508A, and T1067A, were frequent, and one, C445A, was only detected in one out of 40 individuals. Allele-specific polymerase chain reaction as well as cloning of FUT3 alleles showed that the 202 and 314 mutations were co-located on the same allele. COS7 cells transfected with an allele having the 202/314 mutations lacked enzyme activity. Polymerase chain reaction-cleavage assays were established for the genotyping of healthy individuals as well as 20 genuine Lewis-negative cancer patients and 10 non-genuine. The latter have Lewis-negative erythrocytes but saliva ␣1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3 alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower ␣1-4FucT activity in saliva than did homozygous wild-type individuals. The lower enzyme level in heterozygous individuals resulted in a significantly (p < 0.04) lower level of circulating sialyl-Lewis a structure in serum. This has the clinical impact that cut-off levels in tumor marker assays should be defined on the basis of genotyping. In the group of non-genuine Lewis-negative cancer patients, whose erythrocytes convert from Lewis-positive to Lewis-negative during the disease, FUT3 heterozygosity was significantly (p < 0.05) more common.

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Plasma fibronectin (cold insoluble globulin) is a multiple domain glycoprotein composed of two nearly identical disulphide bridged polypeptide chains with molecular weight of 220,000. It is a substrate for factor XIIIa and binds to gelatin and heparin. After digestion of bovine fibronectin with plasmin, four fragments with Mr 29,000, 170,000, 23,000 and 6,000 have been isolated. The N-terminal 29,000 Mr fragment (259 residues) has 10 disulphide bridges within five mutually homologous domains, called “fingers”. The sequence <Gln-Ala-Gln-Gln-Ile-Val-Gln-Pro-Gln- contains the acceptor site for factor XIIIa at position 3. The 170,000 Mr fragment contains both the gelatin and the heparin binding site. After further digestion with chymotrypsin a fragment (Mr 45,000) which binds to gelatin, and a fragment (Mr 30,000) which binds to heparin, have been isolated. The Mr 45,000 fragment consists of at least one more “finger” plus two other mutually homologous domains each with two disulphide bridges. The 23,000 Mr fragment (178 residues) consists of three “finger“- domains and has an N-terminal sequence Val-Arg-. The Mr 6,000 (C-terminal fragment) is a dimer of two identical 26-residue peptides linked by two disulphide bridges. 820 of the expected approx. 1800 residues have been placed in sequence.