Karna Skorstengaard

Amino acid sequence of the factor XIIIa acceptor site in bovine plasma fibronectin

Blood coagulation factor XIII is a proenzyme which can be activated by thrombin [I] to the transglutaminase factor XIII, [2%4]. Factor XIII, catalyzes the formation of e(y-glutamyl)lysyl amide bonds between pairs of y-chains in aggregated fibrin, resulting in its transformation to a highly stable and insoluble covalently cross-linked clot (reviewed in [S-7]). Two other plasma proteins cYz-macroglobulin and fibronectin contain acceptor sites for factor XIII, as shown by incorporation of dansylcadaverine [8]. Only fibronectin, but not aa-macroglobulin, was shown to be crosslinked to fibrin [8]. Cross-linking of fibronectin to collagen [9 ,I 0] and to StaphyZoccocus aureus cells [ 1 l] has been demonstrated. It has also been suggested that rYa-antiplasmin could be covalently linked to. fibrin in a Ca’+dependent reaction probably catalyzed by factor XIII, [ 121. Here we report the ammo acid sequence in bovine fibronectin which contains the glutamine residue labelled with radioactive putrescine by factor XIII,. This glutamine is located at position 3 from the N-terminus of fibronectin.

Influence of Lewis α1-3/4-L-Fucosyltransferase (FUT3) Gene Mutations on Enzyme Activity, Erythrocyte Phenotyping, and Circulating Tumor Marker Sialyl-Lewis a Levels

Fucosylated glycoproteins carrying α1-4 fucose residues are of importance for cell adhesion and as tumor markers. The Lewis gene, FUT3, encodes the only known α1-4-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 α1-4FucT activity. The genuine Lewis-negative individuals had mutations on both FUT3alleles. In 66 healthy individuals, a gene dosage effect was detected as FUT3 heterozygous individuals had a lower α1-4FucT 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.

Sequence location of a putative transglutaminase cross-linking site in human vitronectin

We attempted to locate the glutamine residue in human vitronectin, susceptible to cross‐linking by transglutaminases. Vitronectin was incubated with 14C‐labelled putrescine and plasma factor XIIIa and, after reduction and alkylation, the vitronectin was digested with trypsin. HPLC of the digest followed by scintillation counting revealed one major and two minor radioactivity labelled peaks. Sub‐digestion with Staphylococcus aureus protease, sequence analysis and mass‐spectrometry of the resulting peptides demonstrated that Gln‐93 of vitronectin had incorporated putrescine. Additionally, Gln‐73, Gln‐84 and Gln‐86 were found to be minor sites for incorporation.

Amino acid sequence of a peptide from bovine plasma fibronectin containing a free sulfhydryl group (cysteine).

Plasma fibronectin is a glycoprotein which consists of 2 disulphide-bridged polypeptide chains o fM r 215 000 and 220 000, respectively [ 1,2], whereas fibronectin associated with the cell surface is found both as a dimer and as a multimer [3]. Fibronectin forms strong non-covalent bonds with collagen [4] and heparin [5]. It interacts with cell surfaces [6] and has been reported to bind some other macromolecules including actin [7] and fibrin [8]. Fibronectin can be covalently crosslinked by plasma transglutaminase (factor Xllla) [9] to fibrin [10], collagen [11 ], Staphylococcus aureus cells [12] and to itself [ 13 ]. Cellular fibronectin agglutinates sheep erythrocytes [14] and restores normal fibroblastic morphology to transformed cells [ 14]. Plasma fibronectin is necessary for opsonization of gelatin-coated particles [15]. Degradation of fibronectin with chemicals or proteolytic enzymes has revealed a domain structure (reviewed in [ 16]) with different domains taking part in crosslinking by factor XIII a [ 10,17,18], binding to cells [19], collagen [20] and heparin [19,21 ]. Fibronectin contains 1 -2 thiol groups/monomer [ 16,22-25], and blockage of the free sulfhydryl group prevents binding to cells [22]. Cleavage by S-cyanylation [25] suggests a cysteine located 170 000 mass units from the N-terminus. After digestion of bovine fibronectin with plasmin, 4 fragments o f M r 29 000 (29k), 170 000 (170k), 23 000 (23k) and 6000 (6k) have been isolated [26] and here we report the amino acid sequence of a stretch of 27 amino acid residues of bovine fibronectin

LEWIS ANTIGEN MEDIATED ADHESION OF FRESHLY REMOVED HUMAN BLADDER TUMORS TO E-SELECTIN

PURPOSE Twenty fresh surgical specimens of human bladder tumors were tested for their ability to adhere to recombinant P and E-selectin. The adhesion was correlated to immunological detection of carbohydrate structures. MATERIALS AND METHODS A static titertray assay with immobilized selectins and appropriate controls was used for bladder tumor cell adhesion. On the same tumors expression of carbohydrate structures was examined by immunohistochemistry and Western blotting. RESULTS No tumor bound to P-selectin. Nine tumors showed a high number of cells binding to E-selectin, 5 showed intermediate binding, and 6 showed only rare binding. The specificity of the binding was verified by inhibition with EDTA, by blocking antibodies to E-selectin, and by an acrylamide based sLe(x) (Galbeta1-4 [Fucalpha1-3]GlcNAc-) polymer. The binding was significantly more frequent (p <0.045) in superficial tumors than in invasive tumors. The binding property was correlated to the detection of carbohydrate structures in Western blots and tissue sections of the same tumors, using six different monoclonal antibodies: anti-sLe(a), anti-sLe(x), anti-Le(a), anti-Le(x) (two different clones) and anti-Le(b). Most blot-stainings were smeared indicating a mucin-type carrier molecule, but 115, 55 and 40 kDa bands carrying Le(a) and/or Le(b) epitopes were present in all tumors that bound. The Le(a) structure, as detected by blotting, was the only structure necessary for binding in the center of the wells (p <0.001), and was correlated to number of bound cells (p <0.006). A weaker correlation was found between Le(b) and number of bound cells (p <0.032), whereas it was remarkable that no correlation was found with Le(x) or sLe(x). Immunohistological staining of Le(a) on cell membranes correlated with frequent binding (p <0.003), whereas no correlation was found to secretor and Lewis genotypes. CONCLUSIONS These data on clinical specimens indicate that Lewis antigen mediated E-selectin adhesion may play a role in the human bladder cancer disease.