Determination of Disulfide Bond Assignment of Human Vitamin K-dependent γ-Glutamyl Carboxylase by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Tie, Jian Ke; Mutucumarana, Vasantha P.; Straight, David L.; Carrick, Kevin; Pope, R. Marshall; Stafford, Darrel W.

doi:10.1074/jbc.m309164200

Cited by 34 publications

(64 citation statements)

References 46 publications

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“…Therefore, with the exception of the experiment using 18 O labeling, freshly purified GGCX was deglycosylated before being subjected to in-gel protease digestion (31). After deglycosylation, GGCX samples were fractionated by 10% SDS-PAGE and protein bands were visualized by Coomassie staining.…”

Section: In-gel Protease Digestion and Deglycosylation Of Ggcx For Mamentioning

confidence: 99%

“…After deglycosylation, GGCX samples were fractionated by 10% SDS-PAGE and protein bands were visualized by Coomassie staining. The GGCX band was excised, and the gel pieces were treated as described previously (31). The dry gel pieces were re-hydrated by the addition of (10 ng/μL) protease (trypsin, Endoproteinase Glu-C, or chymotrypsin) in 25 mM NH 4 HCO 3 solution and incubated on ice for 30 minutes.…”

Section: In-gel Protease Digestion and Deglycosylation Of Ggcx For Mamentioning

confidence: 99%

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Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

Tie¹,

Zheng²,

Pope³

et al. 2006

Biochemistry

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The vitamin K-dependent carboxylase is an integral membrane protein which is required for the post-translational modification of a variety of vitamin K-dependent proteins. Previous studies have suggested carboxylase is a glycoprotein with N-linked glycosylation sites. In the present study, we identified the N-glycosylation sites of carboxylase by mass spectrometric peptide mapping analyses combined with site-directed mutagenesis. Our mass spectrometric results show that the N-linked glycosylation in carboxylase occurs at positions N459, N550, N605, and N627. Eliminating these glycosylation sites by changing asparagine to glutamine caused the mutant carboxylase to migrate faster in SDS-PAGE gel analyses, adding further evidence that these sites are glycosylated. In addition, the mutation studies identified N525, a site not recoverable by mass spectroscopy analysis, as a glycosylation site. Furthermore, the potential glycosylation site at N570 is glycosylated only if all the five natural glycosylation sites are simultaneously mutated. Removal of the oligosaccharides by glycosidase from wild-type carboxylase or by eliminating the functional glycosylation sites by site-directed mutagenesis did not affect either the carboxylation or epoxidation activity when the small pentapeptide FLEEL was used as substrate, suggesting that N-linked glycosylation is not required for the enzymatic function of carboxylase. In contrast, when site N570 and the five natural glycosylation sites were mutated simultaneously, the resulting carboxylase protein was degraded. Our results suggest that N-linked glycosylation is not essential for carboxylase enzymatic activity but it is important for protein folding and stability.The vitamin K-dependent carboxylase, also known as gamma-glutamyl carboxylase (GGCX) 1 , is an integral membrane protein of the endoplasmic reticulum. It catalyzes the post-translational modification of specific glutamic acid residues of vitamin K-dependent proteins to γ-carboxyglutamic acid residues. This post-translational modification is critical for the biological functions of the vitamin K-dependent proteins involved in blood coagulation, bone metabolism, signal transduction, and cell proliferation (1-3). In addition to its vitamin K-dependent substrate, the carboxylation reaction, which occurs in the lumen of the ER (4, 5), requires the co-substrates carbon dioxide, oxygen, and vitamin K hydroquinone. During the process of carboxylation, the γ-proton of the glutamic acid is abstracted, followed by the addition of carbon dioxide (6-9). Concomitant with carboxylation, vitamin K hydroquinone is oxidized to vitamin K epoxide which must be converted back to vitamin K by the enzyme vitamin K epoxide reductase, thus completing the vitamin K cycle (1, 10). The formation of vitamin K epoxide during the carboxylation reaction has been called an epoxidation reaction (9,11,12 It has been reported that GGCX binds to lectin suggesting that it is a glycoprotein (14, 15). Moreover, Endoglycosidase H treatment of GGCX purifi...

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Section: In-gel Protease Digestion and Deglycosylation Of Ggcx For Mamentioning

confidence: 99%

Section: In-gel Protease Digestion and Deglycosylation Of Ggcx For Mamentioning

confidence: 99%

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

Tie¹,

Zheng²,

Pope³

et al. 2006

Biochemistry

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“…Previously, we presented evidence that there is a disulfide bond in carboxylase between cysteine residues 99 and 450 (7,13). However, there is some disagreement about whether the disulfide exists (34).…”

Section: The Two-chain Carboxylase Is Joined By a Disulfide Bondmentioning

confidence: 99%

“…In our later work using recombinant proteins, we confirmed that a disulfide bond exists in the one-chain carboxylase. In that study using mass spectrometric analyses, we identified the residues involved in the disulfide as C99 and C450 (13). Also in this study we changed the only two cysteines, other than C450 in the carboxy-terminal peptide, to alanine (C598/700A) and then trypsin-cleaved this carboxylase (13).…”

Section: The Two-chain Carboxylase Is Joined By a Disulfide Bondmentioning

confidence: 99%

“…Approximately 72% and 14% of carboxylase's sequence resides in the ER lumen and cytoplasm, respectively. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry results showed that there is a disulfide bond between cysteine residues 99 and 450 which is important for carboxylase folding and maturation (13). However, there is some disagreement on whether this disulfide bond exists in active carboxylase (14).…”

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Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase

et al. 2008

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We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent γ-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX. We employed this molecule to investigate formation of the one disulfide bond in carboxylase, the transmembrane structure of carboxylase, and the potential interactions among the carboxylase's transmembrane domains. Our results indicate that the two peptides of the two-chain carboxylase are joined by a disulfide bond. Proline 378 is important for the structure necessary for disulfide formation. Results with the P378L carboxylase indicate that noncovalent bonds maintain the two-chain structure even when the disulfide bond is disrupted. As we had previously proposed, the fifth transmembrane domain of carboxylase is the last and only transmembrane domain in the C-terminal peptide of the two-chain carboxylase. We show that the noncovalent association between the two chains of carboxylase involves an interaction between the fifth transmembrane domain and the second transmembrane domain. Results of a homology model of transmembrane domains 2 and 5 suggest that not only do these two domains associate but that transmembrane domain 2 may interact with another transmembrane domain. This latter interaction may be mediated at least in part by a motif of glycine residues in the second transmembrane domain.The vitamin K-dependent γ-glutamyl carboxylase is a 758-residue integral membrane glycoprotein of the endoplasmic reticulum (ER). 1 It catalyzes the posttranslational modification of specific glutamic acid residues of vitamin K-dependent proteins to γ-carboxyglutamic acid residues. This posttranslational modification is critical for the biological † This work was supported by National Institutes of Health (NIH) Grant HL48318 (to D.W.S.) and by the Intramural Research Program of the NIH and NIEHS (to L.P.).*Author to whom correspondence should be addressed. Phone: 919-962-2267. Fax: 919-962-9266. jktie@email.unc.edu. ‡ University of North Carolina at Chapel Hill. § National Institute of Environmental Health Sciences, NIH.1 Abbreviations: ER, endoplasmic reticulum; TMD, transmembrane domain; NEM, CHAPS,dimethylammonio]-1-propanesulfonate; DTT, dithiothreitol; MOPS, 3-(N-morpholino)propanesulfonic acid (4-morpholinepropanesulfonic acid); MES, 2-(N-morpholino)ethanesulfonic acid (4-morpholineethanesulfonic acid); vitamin K 1(20) , 2-methyl-3-phytyl-1,4-naphthoquinone; PNGase F, peptide:N-glycosidase F; proFIX, 19 amino acid peptide comprising residues TVFLDHENANKILNRPKRY of human profactor IX; SRII, sensory rhodopsin II; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride.

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Application of MALDI TOF/TOF mass spectrometry and collision‐induced dissociation for the identification of disulfide‐bonded peptides

Janecki¹,

Nemeth²

2011

J. Mass Spectrom.

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This paper describes a method for the fast identification and composition of disulfide-bonded peptides. A unique fragmentation signature of inter-disulfide-bonded peptides is detected using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF)/TOF mass spectrometry and high-energy collision-induced dissociation (CID). This fragmentation pattern identifies peptides with an interconnected disulfide bond and provides information regarding the composition of the peptides involved in the pairing. The distinctive signature produced using CID is a triplet of ions resulting from the cleavage of the disulfide bond to produce dehydroalanine, cysteine or thiocysteine product ions. This method is not applicable to intra-peptide disulfide bonds, as the cleavage mechanism is not the same and a triplet pattern is not observed. This method has been successfully applied to identifying disulfide-bonded peptides in a number of control digestions, as well as study samples where disulfide bond networks were postulated and/or unknown.

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Determination of Disulfide Bond Assignment of Human Vitamin K-dependent γ-Glutamyl Carboxylase by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

Cited by 34 publications

References 46 publications

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

Identification of the N-Linked Glycosylation Sites of Vitamin K-Dependent Carboxylase and Effect of Glycosylation on Carboxylase Function

Transmembrane Domain Interactions and Residue Proline 378 Are Essential for Proper Structure, Especially Disulfide Bond Formation, in the Human Vitamin K-Dependent γ-Glutamyl Carboxylase

Application of MALDI TOF/TOF mass spectrometry and collision‐induced dissociation for the identification of disulfide‐bonded peptides

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