Methylation of γ-Carboxylated Glu (Gla) Allows Detection by Liquid Chromatography–Mass Spectrometry and the Identification of Gla Residues in the γ-Glutamyl Carboxylase

Hallgren, Kevin W.; Zhang, D.; Kinter, Michael; Willard, Belinda; Berkner, Kathleen L.

doi:10.1021/pr3003722

Cited by 23 publications

(43 citation statements)

References 23 publications

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“…GGCX carboxylation was reduced as early as e18.5 and was undetectable at P7 using the immunoprecipitation assay. In agreement with this and previous studies in cells (14,15), we also observed in total liver extracts (i.e., input) that GGCX migrates faster in the absence of Vkorc1 because of its incomplete carboxylation. Therefore, carboxylation is reduced in Vkorc1 -/-mice between the pre-and postnatal period but is not completely blunted, suggesting that a second enzyme supports KO reduction during these periods. )…”

Section: Carboxylated Vkd Proteins Are Present In Vkorc1supporting

confidence: 93%

VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo

et al. 2018

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Section: Carboxylated Vkd Proteins Are Present In Vkorc1supporting

confidence: 93%

VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo

et al. 2018

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“…). Consequently, these neutral losses make identification of the modified peptides more difficult . CO 2 loss during fragmentation gives b‐ or y‐ion series consistent with the unmodified protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

Influence of carboxylation on osteocalcin detection by mass spectrometry

Cleland

Thomas

Gundberg

et al. 2016

Rapid Comm Mass Spectrometry

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Rationale Osteocalcin is a small, abundant bone protein that is difficult to detect using high-throughput MS/MS proteomic approaches from bone protein extracts, and is predominantly detected by non-MS immunological methods. Here, we analyze bovine osteocalcin and its post-translational modifications to determine why a protein of this size goes undetected. Methods Osteocalcin was purified from cow bone using well-established methods. Intact osteocalcin or trypsin digested osteocalcin were separated using an Agilent 1200 series HPLC and analyzed using a ThermoScientific LTQ-Orbitrap XL after fragmentation with higher-energy collision dissociation. Data were analyzed using Mascot or Prosight Lite. Results Our results support previous findings that the cow osteocalcin has up to three carboxylations using both intact osteocalcin and digested forms. Using Mascot, we were able to detect osteocalcin peptides, but no fragments that localized the carboxylations. Full annotation using Prosight Lite of the intact (three carboxylations), N-terminal peptide (one carboxylation), and middle peptide (two carboxylations) showed complete fragmentation was present, but complete neutral loss was observed. Conclusions Osteocalcin carboxylation, and its associated neutral losses, makes high-throughput detection of this protein challenging; however, alternative fragmentation or limited purification can overcome these challenges.

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“…We utilized a previously described methodology specifically for identifying the location of γ-carboxylation on proteins [11]. Due to the instability of γ-carboxyglutamate and the interference of its highly negative charge with ionization, methylation was performed to both eliminate the negative charge and protect the carboxyl group from neutral loss.…”

Section: Discussionmentioning

confidence: 99%

Vitamin K epoxide reductase regulation of androgen receptor activity

et al. 2017

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Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.

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Methylation of γ-Carboxylated Glu (Gla) Allows Detection by Liquid Chromatography–Mass Spectrometry and the Identification of Gla Residues in the γ-Glutamyl Carboxylase

Cited by 23 publications

References 23 publications

VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo

VKOR paralog VKORC1L1 supports vitamin K–dependent protein carboxylation in vivo

Influence of carboxylation on osteocalcin detection by mass spectrometry

Vitamin K epoxide reductase regulation of androgen receptor activity

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