RESUMO
Tissue factor (TF), a membrane protein, is an initiator of blood coagulation in vivo. In this review we discuss how posttranslational modifications affect activity and other properties of TF. Glycosylation of the extracellular domain and the composition of carbohydrates at three glycosylation sites have an influence on TF activity in the extrinsic FXase by increasing the rate of FX proteolysis. No influence of TF glycosylation on the activity of the FVIIa/TF complex towards small synthetic substrates was observed, suggesting that glycosylation has no effect on TF interaction with FVIIa. There are no published data suggesting a direct influence of phosphorylation or palmitoylation in the cytoplasmic domain on TF procoagulant activity. There has been a debate in the recent literature related to the role and formation of the Cys¹86-Cys²°9 disulfide bond. Published opinions from various laboratories range from this bond being essential for the expression of cell TF activity to having no role in it. Overall, it is clear that some modifications of TF have an effect on TF procoagulant activity, signaling functions and trafficking. The influences of other modifications are debatable.
Assuntos
Processamento de Proteína Pós-Traducional , Tromboplastina/metabolismo , Animais , Dissulfetos/metabolismo , Glicosilação , Humanos , Ácido Palmítico/metabolismo , FosforilaçãoRESUMO
We determined the pattern of cross-reactivity of a panel of anti-streptokinase (SK) monoclonal antibodies (mAbs) with SK variants in order to map the antigenic and functional epitope of SK. Comparison of the pattern of cross-reactivity of the anti-SK mAb A4.3 with SK variants and sequence alignments of SK variants and native (n) SK suggested that mutation of Ser 138 to Lys results in loss of binding of mAb A4.3 to SK variants. However, this mutation does not affect formation of activator complex by these proteins. The epitope specificity of the mAb A4.3 was further confirmed by mutating Ser 138 to Lys in n SK. Monoclonal Ab A4.3 did not bind to mutant SK (Ser138Lys). Activator activity of mutant SK (Ser138Lys) was indistinguishable from that of n SK and recombinant n SK. Since addition of A4.3 mAb to an equimolar mixture of SK and human plasminogen inhibits activator complex formation, the sequences spanning position 138 are likely important for formation of streptokinase-plasminogen activator complex or processing of the plasminogen substrate.
Assuntos
Antígenos de Bactérias/química , Estreptoquinase/química , Estreptoquinase/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Antígenos de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Domínio Catalítico/genética , Reações Cruzadas , DNA Bacteriano/genética , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Genes Bacterianos , Variação Genética , Humanos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasminogênio/química , Plasminogênio/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Serina/química , Streptococcus/enzimologia , Streptococcus/genética , Estreptoquinase/genética , Estreptoquinase/metabolismoRESUMO
To select sequences complementary to their binding sites, two anti-streptokinase (SK) monoclonal antibodies (mAbs), A4.5 and A5.5, were used in biopanning of 15-mer and hexamer phage-displayed peptide libraries, respectively. mAb A4.5 inhibits the catalytic activity of streptokinase-plasminogen activator complex (SKPAC), the binding of plasminogen to SK and the binding of human anti-SK polyclonal Abs to SK. All clones selected from the 15-mer peptide library by mAb A4.5 had identical nucleotide and amino acid sequences, RSVYRCSPFVGCWFG. An 11-mer peptide (peptide A4.5, YRCSPFVGCWF) derived from this sequence inhibited the binding of mAb A4.5 and human anti-SK polyclonal Abs to SK as well as the catalytic activity of both SKPAC and plasmin. The binding of the second mAb (mAb A5.5) to SK is lost upon interaction of SK with plasminogen, suggesting that sequences selected by this mAb are likely associated with the C-terminal cleavage site of SK. Biopanning of a hexamer peptide library with mAb A5.5 selected the sequence RYLQDY that is homologous to residues 324-328, adjacent to one possible C-terminal cleavage site in SK. A 10-mer synthetic peptide (LDFRDLYDPR) corresponding to residues 321-330 in SK specifically inhibited the binding of mAb A5.5 to SK. The selection and characterization of these two peptides enhances our understanding of SK structure, maps an antigenic epitope, and identifies a peptide inhibitor of plasminogen activation.
