Abstract
Factor X is a plasma protein involved in both the intrinsic and extrinsic pathways of blood coagulation. Post-translational modifications of the protein involve gamma-carboxylation of specific glutamic acid residues, beta-hydroxylation of one aspartic acid residue, and N- and O-linked glycosylation. Even though it is known that gamma-carboxylation is instrumental in regulating biological activity, the role of glycosylation in the function and properties of factor X has not been previously investigated. We utilized lectin binding and glycosidase treatment to investigate the functional role of carbohydrates on the activation peptide of factor X. Sambucus nigra agglutinin, a lectin that binds to sialic acid terminally linked alpha(2-6) to galactose or N-acetyl-galactosamine inhibits activation of human factor X in a dose-dependent manner. Inhibition of activation was observed for both intrinsic (factor IXa/VIIIa) and extrinsic (factor VIIa/tissue factor) pathway complexes. In accordance with this, selective removal of sialic acid residues on the activation peptide of factor X by neuraminidase also results in a drastic reduction of activation of the zymogen by these complexes. Corresponding reduction of activity in classical clotting assays (activated partial thromboplastin time and prothrombin time) also agrees with this observation. These results suggest a possible role of N-linked carbohydrates in the activation of factor X.
Highlights
Factor X is a plasma protein involved in both the factor by either the intrinsic or extrinsic pathway (3)
We utilized lectin binding and glycosidase treatment to investigate the functional role of carbohydrates on the activation peptide of factor X
Selectivreemovalof sialic acid residues on the activation peptide of factor X by neuraminidase results in a drastic reduction of activa
Summary
By SNA-Human or bovine factor X (1pM) was incubated bti4bcrwoltHPraodgiiineohona)inbrkdNdeund-alavCdseeereaNmd0utANeieli.cheonydns-cSr-t-aasogrgvaeeoegdNo,rintbaclattsdnaloofywaelfyetAedtcSdaeaaichnctciothocsiociyDbtduaeodcntiuntrlirnsomsnooaSegnismiinNssoztrnadd-ldaees,aoautPal-bian.sswircwttacnocAniretapcsooSeoivebdfletu(Gfscnaslraiihita1yugneoancncyEc-emamigNcPodtld3ctroagnfelNos-mi)ainraifhrani-trrnCzrXaaeAldhNceaueXraeklcsaeseWrimic,e-itaiericttdtaoibwdzbydbnadhonaecucrieulouchdsnesonsgeedoeohit"aitmtsdldscrhecmuyyuniXn.ihoirnlpdbtclnogooingrDloronagrhtTtceteaabaesevesSxatlaalctnr.tiueatAoobdnomomomeadScnrtielgdcnMXettsNiheiaiconhintcncdfnunaouiAsoueouaaznimhgaIrnifcgmsle.oseymb.lstnisatLoisyobtaeGoniwitneihnerztdlnreun0"sNhiiseeelicnnde.-fsyrepstAgfadekoaiSrtaNscglnieyecn,cuycihtntav-dtwsbmpoootFbc0uliguioieaarvroahs-macintlber(clgatunhyicaXdj2aitcatlsestcreotrXid-ihyonlbuca.anos6yicrtroatenogsnh.)feaebe,haasDrtedPndsiXToccypanuti(sStcv--atbhhe1d.tolAhynty-poeesrsmad- -oswcvtoplGsnTgoXoeiSositpffroilavooDhilgsyeeEytntnetfSonuedihchcScaftrconaNoiiwafonoc-afdvitnllesiPisfnyitliAauacdncinzoig.cAdtadyrtareisorhoiuaiccalOdaGottntlXctsasbteoywbfsogeieubtEadlivites-morywemSrntuta-aniedrSuecdpnpoeaCtmaeceuluinilrynaunaomeuneooerobplytctgrcxioneybaotaunrtcaaneehmdiesittumocgbvisliidieztitn.vnaeloszhiieuefcitstedvnnFesafahcosRsfdiashiicttoienetNdgioeitewetifaocte.afmhootdr-ltandBiasuroiln1hStocceioksXmhllbsunNvitwuesbeaniyemtk.ahplaevsAcserteTxleolntebhiearascahpddhweoartoet(ntceefifvuia0tgretsaaovamri.heaslln5iSscennayties,tmeh-tndmiaciuidctro2nodneosheepgti0routngsismnetea;dncXeyuhsbtprWouoss(lstneoiitahdssvecnRnMitivtepseSixasadaotidiene-o)erslptofneNpsdidoMiasernfeecinrohcfaevAdcoabohoflsAunmpseutogffteri,uemiosereaeAcmrtfethmdunntetsoceat,iu,geisdtrashnpagnososebdma,enSaghnhiyrh.rrnvdtfueiNaotfDAfarettsnXnaSbwaaescAliSeilcoyciesptntnifnnxtAoitqemeag4oahdod)dtlmruccrfor.iureiod"iottbinX0wefenoaCeenitXiigddssosrsrc-.--s. nic. T o galactose and binds to both bovine and human factor X. No lectin binding was observed to the light chain for either human or bovine factors X or Xa. I factor complex. Aliquots of human or bovine factor X were activated using factors. Bovine factor X withthe various glycosidases led to This moremodesteffect may be accounted for by the less alterations in theelectrophoretic mobilities of the heavy than complete removal of all N-linked sugars under the nonchains of theproteins (Fig. 2). Mobility was detected on the light chainOs.vernight digestion The relative activities of the variousglycosidase-treated with glycosidases were deemedsufficient,sinceuncleaved human and bovine factors in two plasma clotting assays are heavy chain was no longer visible on Coomassie Bluestaining shown inTable 11. The effects on the extent of activation of the deglycosylated proteins obtained
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