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Titolo:
THE FUNCTIONS OF THE FIRST EPIDERMAL GROWTH-FACTOR HOMOLOGY REGION OFHUMAN PROTEIN-C AS REVEALED BY A CHARGE-TO-ALANINE SCANNING MUTAGENESIS INVESTIGATION
Autore:
CHENG CH; GENG JP; CASTELLINO FJ;
Indirizzi:
UNIV NOTRE DAME,DEPT CHEM & BIOCHEM NOTRE DAME IN 46556 UNIV NOTRE DAME,DEPT CHEM & BIOCHEM NOTRE DAME IN 46556
Titolo Testata:
Biological chemistry
fascicolo: 12, volume: 378, anno: 1997,
pagine: 1491 - 1500
SICI:
1431-6730(1997)378:12<1491:TFOTFE>2.0.ZU;2-Q
Fonte:
ISI
Lingua:
ENG
Soggetto:
FACTOR-LIKE DOMAIN; THROMBIN-THROMBOMODULIN COMPLEX; COAGULATION FACTOR-IX; INVITRO ANTICOAGULANT ACTIVITY; ACIDIC PHOSPHOLIPID-VESICLES; AFFINITY CA2+-BINDING SITE; CALCIUM-BINDING SITE; EGF-LIKE DOMAINS; TISSUE FACTOR; COFACTOR ACTIVITY;
Keywords:
CALCIUM BINDING; MONOCLONAL ANTIBODIES; PROTEIN C; PROTEIN DOMAINS; PROTEIN MUTAGENESIS; PROTEIN PROCESSING; SERINE PROTEASES;
Tipo documento:
Article
Natura:
Periodico
Citazioni:
56
Recensione:
Indirizzi per estratti:
Citazione:
C.H. Cheng et al., "THE FUNCTIONS OF THE FIRST EPIDERMAL GROWTH-FACTOR HOMOLOGY REGION OFHUMAN PROTEIN-C AS REVEALED BY A CHARGE-TO-ALANINE SCANNING MUTAGENESIS INVESTIGATION", Biological chemistry, 378(12), 1997, pp. 1491-1500

Abstract

Variant proteins containing charge-to-alanine mutations of single amino acid residues and clusters of such groups contained in the epidermal growth factor 1 (EGF1) homology unit of human protein C (PC) have been accomplished, resulting in the following recombinant (r) mutant proteins: r-[E(56)A/H(57)A]PC; r-[H(66)A]-PC; r-[D(71)A]PC; r-[D(79)A/R(81)A]PC; r-[E(85)A/R(87)A]PC; and r-[R(91)A/E(92)A]PC. Studies of the mutant proteins with a variety of Ca2+-dependent and Ca2+-independent monoclonal antibodies not only led to identification of the epitopes ofthese antibodies, but also confirmed the importance of D/beta-hydroxyaspartic acid (Hya)(71) as one probable coordination site for Ca2+. Employing these antibodies, it was also revealed that Ca2+ binding to its site in the EGF1 region of PC did not influence Ca2+ binding or adoption of the Ca2+-dependent conformation of the gamma-carboxyglutamic acid domain of this same protein, In addition, the Ca2+-induced inhibition of PC activation by thrombin, and the kinetic constants for activation of PC by the thrombin/thrombomodulin complex, were only modestly affected by any of the mutations. The mutants r-[E(56)A/H(57)A]APC andr-[H(66)A]APC displayed at least 70% of wild type r-APC activity in afVIII inactivation assay, while r-[D(79)A/R(81)A]APC, r-[E(85)A/R(87)A]APC and r-[R(91)A/E(92)A]APC possessed only approximately 40% activity in that same assay. The special role of D/Hya(71) in this process was confirmed by showing that r-[D(71)A]APC was inactive in the fVIII-inactivation assay, These findings demonstrate that some of the chargedresidues of EGF1, most notably those in the carboxyterminal region ofthis domain, participate as partial determinants of the anticoagulantactivity of APC. Overall, with the exceptions noted, the data generally suggest that the charged residues of the EGF1 domain of PC, and theCa2+ binding site contained within this module, are likely more involved with maintenance of the overall structural integrity of this module rather than with its direct functional interactions with effecters, activators, or substrates of PC and APC. Lastly, functional Ca2+ binding to the Gla domain of PC is not significantly influenced by the binding of Ca2+ to the EGF1 module.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 23/09/20 alle ore 08:35:37