The Propeptide Binding Site of the Bovine γ-Glutamyl Carboxylase

Abstract

␥-Glutamyl carboxylase is an integral membrane protein required for the posttranslational modification of vitamin K-dependent proteins. The main recognition between the enzyme and its substrates is through an 18-amino acid propeptide. It has been reported that this binding site resides in the amino-terminal third of the ␥-glutamyl carboxylase molecule (Yamada, M., Kuliopulos, A., Nelson, N. P., Roth, D. A., Furie, B., Furie, B. C., and Walsh, C. T. (1995) Biochemistry 34, 481-489). In contrast, we found the binding site in the carboxyl half of the ␥-glutamyl carboxylase. We show that the carboxylase may be cleaved by trypsin into an amino-terminal 30-kDa and a carboxyl-terminal 60-kDa fragment joined by a disulfide bond(s), and the propeptide binds to the 60-kDa fragment. The sequence of the amino terminus of the 60-kDa fragment reveals that the primary trypsin-sensitive sites are at residues 349 and 351. Furthermore, the tryptic fragment that cross-links to the propeptide also reacts with an antibody specific to the carboxyl portion of the ␥-glutamyl carboxylase. In addition, cyanogen bromide cleavage of bovine ␥-glutamyl carboxylase cross-linked to the peptide comprising residues TVFLDHENANKILNRPKRY of human factor IX yields a cross-linked fragment of 16 kDa from the carboxyl half of the molecule, the amino-terminal sequence of which begins at residue 438. Thus, the propeptide binding site lies carboxyl-terminal to residue 438 and is predicted to be in the lumen of the endoplasmic reticulum.␥-Glutamyl carboxylation, accomplished by the enzyme ␥-glutamyl carboxylase, is a posttranslational modification essential for the biological activities of a number of vitamin K-dependent proteins. The importance of ␥-glutamyl carboxylation is demonstrated by the various functions of the vitamin K-dependent proteins. The best characterized functions are related to coagulation and are exemplified by proteins such as prothrombin, factor VII, factor IX, factor X, protein C, and protein S. In addition, there are two bone proteins, osteocalcin and matrix Gla protein (1), and the newly discovered growth arrest-specific protein Gas 6 (2).The ␥-glutamyl carboxylation reaction occurs in the endoplasmic reticulum (3-5), where the enzyme ␥-glutamyl carboxylase uses the small substrates carbon dioxide, oxygen, and vitamin K hydroquinone to convert specific glutamic acid residues of the vitamin K-dependent protein into the ␥-carboxyl glutamic acid Gla. During the process of ␥-glutamyl carboxylation, the vitamin K hydroquinone is converted to vitamin K epoxide, which must be recycled to vitamin K hydroquinone by the enzyme epoxide reductase for the reaction to continue.The enzymatic activity of the ␥-glutamyl carboxylase was first discovered in the postmitochondrial supernatant of hepatocytes (6). Numerous early studies used microsomes as the crude enzyme sources and the pentapeptide FLEEL as the in vitro substrate to establish a basic understanding of ␥-glutamyl carboxylation (7). By comparing the cDNA-deduced amino acid sequences of di...

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

“…Previous work from our laboratory showed that limited trypsin digestion of GGCX results in a disulfide-linked 30 kDa amino-terminal and 60 kDa carboxyl-terminal fragments (16) (Figure 1, lane 3). Figure 1 (lane 4) shows that only the 60 kDa carboxyl-terminal tryptic fragment is sensitive to PNGase F digestion indicating, in agreement with our previous report (16), that N-linked glycosylation occurs only on the 60 kDa carboxyl-terminal tryptic fragment of GGCX. Asparagine 159 is the only potential glycosylation site in the 30 kDa amino-terminal tryptic fragment and these results indicate that it is not glycosylated in vivo.…”

“…Figure 1 demonstrates that PNGase F treated GGCX (lane 2) migrates faster than the untreated sample (lane 1) in SDS-PAGE analysis indicating that GGCX is an N-linked glycoprotein. Previous work from our laboratory showed that limited trypsin digestion of GGCX results in a disulfide-linked 30 kDa amino-terminal and 60 kDa carboxyl-terminal fragments (16) (Figure 1, lane 3). Figure 1 (lane 4) shows that only the 60 kDa carboxyl-terminal tryptic fragment is sensitive to PNGase F digestion indicating, in agreement with our previous report (16), that N-linked glycosylation occurs only on the 60 kDa carboxyl-terminal tryptic fragment of GGCX.…”

Identification of a Drosophila Vitamin K-dependent γ-Glutamyl Carboxylase

“…Nevertheless, a hydrophobic propeptide different from the commonly known mammalian propeptides appears to be present in the substrate of the Conus carboxylase (1, 10). Therefore, one might expect to find a propeptide-binding site on the dGC that is not homologous to the hGC-binding site but that is located in the same region we previously determined to be the location of the mammalian site (11). One major difference between the Drosophila and mammalian ␥-glutamyl carboxylases is that the dGC is 86 amino acids shorter than the human enzyme from the carboxyl terminus.…”

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

“…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).…”

“…On the basis of affinity peptidesubstrate labeling, Yamada et al reported that the propeptide binds to the carboxylase between residues 50 and 225 (6). Propeptide cross-linking experiments from our laboratory suggested that the propeptide binding region of carboxylase is between residues 438 and 507 (7). Further studies employing site-directed mutagenesis showed that at least part of the propeptide binding region includes residues between 495 and 513 of the carboxylase (8).…”