Cloning and Expression of the cDNA for Human γ-glutamyl Carboxylase

Wu, Sheue Mei; Cheung, Wing Fai; Frazier, D; Stafford, Darrel W.

doi:10.1126/science.1749935

Cited by 195 publications

(146 citation statements)

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“…All of the radioactivity was recovered in the gla peak, with the cpm in this peak accounting for 98% of the cpm injected onto the C18 column. Quantitation of gla, asp, and glu peaks by fluorescence indicated an asp-to-glu ratio of 1.3, which would be predicted from the known sequence of the human carboxylase (14). The ratio of gla-to-asp indicated 3 mol of gla per mol of carboxylase.…”

Section: Resultsmentioning

confidence: 99%

“…This cDNA encodes a 95-kDa protein confirmed to be the carboxylase by its ability to effect peptide activity when expressed in baculovirusinfected insect cells, which do not otherwise contain carboxylase activity (15). The cDNA sequence for the carboxylase does not predict any functional domains: A weak homology to lipoxygenases was detected, and some homology to the carboxylase recognition sequence of matrix gla protein (but not to any other VKD protein) was reported (14,16). It was therefore surprising when we discovered that the carboxylase itself is a VKD protein.…”

mentioning

confidence: 99%

“…The carboxylase has been purified from bovine liver and from the human 293 cell line (11)(12)(13), and a cDNA encoding the human carboxylase has been isolated (14). This cDNA encodes a 95-kDa protein confirmed to be the carboxylase by its ability to effect peptide activity when expressed in baculovirusinfected insect cells, which do not otherwise contain carboxylase activity (15).…”

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confidence: 99%

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Vitamin K-dependent carboxylation of the carboxylase

Berkner

Pudota

1998

Proc. Natl. Acad. Sci. U.S.A.

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Vitamin K-dependent (VKD) proteins require modification by the VKD-␥-glutamyl carboxylase, an enzyme that converts clusters of glus to glas in a reaction that requires vitamin K hydroquinone, for their activity. We have discovered that the carboxylase also carboxylates itself in a reaction dependent on vitamin K. When pure human recombinant carboxylase was incubated in vitro with 14 CO 2 and then analyzed after SDS͞PAGE, a radiolabeled band corresponding to the size of the carboxylase was observed. Subsequent gla analysis of in vitro-modified carboxylase by base hydrolysis and HPLC showed that all of the radioactivity could be attributed to gla residues. Quantitation of gla, asp, and glu residues indicated 3 mol gla͞mol carboxylase. Radiolabeled gla was acid-labile, confirming its identity, and was not observed if vitamin K was not included in the in vitro reaction. Carboxylase carboxylation also was detected in baculovirus-(carboxylase)-infected insect cells but not in mock-infected insect cells, which do not express endogenous VKD proteins or carboxylase. Finally, we showed that the carboxylase was carboxylated in vivo. Carboxylase was purified from recombinant carboxylase BHK cells cultured in the presence or absence of vitamin K and analyzed for gla residues. Carboxylation of the carboxylase only was observed with carboxylase isolated from BHK cells cultured in vitamin K, and 3 mol gla͞mol carboxylase were detected. Analyses of carboxylase and factor IX carboxylation in vitro suggest a possible role for carboxylase carboxylation in factor IX turnover, and in vivo studies suggest a potential role in carboxylase stability. The discovery of carboxylase carboxylation has broad implications for the mechanism of VKD protein carboxylation and Warfarin-based anti-coagulant therapies that need to be considered both retrospectively and in the future.Vitamin K-dependent (VKD) proteins undergo an unusual posttranslational modification required for their biological activity, namely the carboxylation of clusters of glu residues to ␥-glutamyl glus, or glas, in a region of the VKD proteins called the gla domain (1, 2). Carboxylation of the VKD proteins effects their Ca 2ϩ -mediated interaction with phospholipid bilayers and is carried out by an integral membrane endoplasmic reticulum enzyme, the VKD-␥-glutamyl carboxylase. The carboxylase modifies VKD substrates by using CO 2 , O 2 , and vitamin K hydroquinone as cofactors, and the carboxylase is also an epoxidase, converting the vitamin K hydroquinone to vitamin K 2,3-epoxide. Subsequent regeneration of the vitamin K hydroquinone cofactor is carried out by a reductase that has been characterized but not yet identified (3, 4). Carboxylation of the gla domain involves the modification of multiple glus, ranging from 3 to 12 for the different VKD proteins. This multiplicity raises the question of whether the carboxylase is a processive enzyme, i.e., effecting all modifications as a result of a single binding event. When purified bovine liver carboxylase was coincubated...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Vitamin K-dependent carboxylation of the carboxylase

Berkner

Pudota

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…La quantité de vitamine K apportée par l'alimentation étant limitée, il est impératif de recycler cet époxyde en vitamine K réduite, à nouveau disponible pour la γ-glutamyl carboxylase, et c'est cette réaction qu'accomplit la VKOR (Figure 1). La fonction de la γ-glutamyl carboxylase [1] est de transformer en acide γ-carboxy-glutamique les neuf à douze glutamates du domaine amino-terminal de cinq proenzymes (les facteurs VII, IX, X, la prothrombine, et la protéine C) et d'un cofacteur (la protéine S), qui, synthétisés par les hépatocytes, sont tous essentiels pour l'hémo-stase ( Figure 2) [2,3]. D'autres protéines, dont le rôle n'est pas toujours bien défini, subissent cette modification post-traductionnelle : la protéine Z également d'origine hépatique, l'ostéocalcine impliquée dans le métabolisme osseux, la protéine anti-apoptotique Gas6 exprimée par de nombreuses cellules, et deux protéines transmembranaires dites «riches en prolines» exprimées dans la moelle épinière et la thyroïde.…”

Section: La Cible De La Warfarine Identifiéeunclassified

“…Toutefois, certains rongeurs résistent à la warfarine [5], et plusieurs cas de résistance ont été décrits chez l'homme [6]; on peut y ajouter trois cas humains de déficience dans les encéphalopathies spongiformes transmissibles, en particulier dans les formes familiales humaines ou au cours de la transmission expérimen-tale de l'encéphalopathie spongiforme bovine. Par ailleurs, et de façon plus remarquable encore, l'accumulation de PrP Sc dans le cerveau n'est pas toujours accompagnée d'une neurodégénéres-cence, comme le confirme un article récent [1]. En revanche, des formes intracytoplasmiques ou transmembranaires dérivées de la protéine cellulaire normale, la PrP C , se sont révélées toxiques pour les cel- > Il apparaît de plus en plus évident que la forme pathologique de la protéine du prion, la PrP Sc , ne peut pas, à elle seule, rendre compte des phénomènes de neurodégénérescence observés dans les maladies à prion.…”

Section: La Cible De La Warfarine Identifiéeunclassified