A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.
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ABSTRACT:
SUBMITTER: Rose K
PROVIDER: S-EPMC1162513 | biostudies-other | 1980 Apr
REPOSITORIES: biostudies-other
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Project description:The detailed mass-spectrometric evidence for our original findings [Magnusson et al. (1974) FEBS Lett. 44, 189-193] of ten gamma-carboxyglutamic acid residues in the N-terminal calcium-binding polypeptide of prothrombin is presented. The identification and sequence location of gamma-carboxyglutamic acid was made by electron-impact and field-desorption studies on acetyl permethyl peptide derivatives, and on the free amino acid. Details of the derivatives formed, and how this new amino acid may be easily recognized and sequenced from the mass spectrum, are given as a basis for future work.
| S-EPMC1172636 | biostudies-other
Project description:Vitamin K-dependent coagulation factors undergo several post-translational modifications before the proteins are secreted into the blood as functional zymogens of the coagulation system. The modifications include Asn-linked glycosylation, Asn/Asp hydroxylation, removal of a signal peptide for translocation of the polypeptide into the endoplasmic reticulum and removal of a propeptide which, when attached to the intracellular coagulation factor precursor, directs the protein for vitamin K-dependent gamma-carboxylation. gamma-Carboxylation of targeted Glu residues results in formation of Ca(2+)-binding gamma-carboxyglutamic acid (Gla) residues. Ca2+ binding by these residues induces a conformational change in the protein which is a necessary event for optimal activation or activity of the clotting factor in blood. In the present study we have monitored the intracellular prothrombin precursor in the secretory pathway of liver cells to determine the effect that the propeptide has on Ca(2+)-dependent folding of the protein. The data provide evidence that the Ca(2+)-induced conformational change required for activation of prothrombin coincides with release of the propeptide in the trans-Golgi apparatus of the liver cell and elucidates an important function for the endoproteinase furin in biosynthesis of vitamin K-dependent clotting factors.
| S-EPMC1132428 | biostudies-other
Project description:Due to its much slower deamidation rate compared to that of asparagine (Asn), studies on glutamine (Gln) deamidation have been scarce, especially on the differentiation of its isomeric deamidation products: alpha- and gamma-glutamic acid (Glu). It has been shown previously that electron capture dissociation (ECD) can be used to generate diagnostic ions for the deamidation products of Asn: aspartic acid (Asp) and isoaspartic acid (isoAsp). The current study explores the possibility of an extension of this ECD based method to the differentiation of the alpha- and gamma-Glu residues, using three human Crystallin peptides (alphaA (1-11), betaB2 (4-14), and gammaS (52-71)) and their potentially deamidated forms as model peptides. It was found that the z(*)-72 ions can be used to both identify the existence and locate the position of the gamma-Glu residues. When the peptide contains a charge carrier near its N-terminus, the c+57 and c+59 ions may also be generated at the gamma-Glu residue. It was unclear whether formation of these N-terminal diagnostic ions is specific to the Pro-gamma-Glu sequence. Unlike the Asp containing peptides, the Glu containing peptides generally do not produce diagnostic side chain loss ions, due to the instability of the resulting radical. The presence of Glu residue(s) may be inferred from the observation of a series of z(n)(*)-59 ions, although it was neither site specific nor without interference from the gamma-Glu residues. Finally, several interference peaks exist in the ECD spectra, which highlights the importance of the use of high performance mass spectrometers for confident identification of gamma-Glu residues.
| S-EPMC2872026 | biostudies-literature
Project description:Two human cDNAs that encode novel vitamin K-dependent proteins have been cloned and sequenced. The predicted amino acid sequences suggest that both are single-pass transmembrane proteins with amino-terminal gamma-carboxyglutamic acid-containing domains preceded by the typical propeptide sequences required for posttranslational gamma-carboxylation of glutamic acid residues. The polypeptides, with deduced molecular masses of 23 and 17 kDa, are proline-rich within their putative cytoplasmic domains and contain several copies of the sequences PPXY and PXXP, motifs found in a variety of signaling and cytoskeletal proteins. Accordingly, these two proteins have been called proline-rich Gla proteins (PRGP1 and PRGP2). Unlike the gamma-carboxyglutamic acid domain-containing proteins of the blood coagulation cascade, the two PRGPs are expressed in a variety of extrahepatic tissues, with PRGP1 and PRGP2 most abundantly expressed in the spinal cord and thyroid, respectively, among those tissues tested. Thus, these observations suggest a novel physiological role for these two new members of the vitamin K-dependent family of proteins.
| S-EPMC23027 | biostudies-literature
Project description:The main forms of poly-γ-glutamic acid (γ-PGA) applied in agriculture include agricultural γ-PGA and γ-PGA super absorbent polymer (SAP). Laboratory experiments were conducted with a check treatment CK (no γ-PGA added) and two different forms of γ-PGA added to sandy loam soil (T and TM stand for γ-PGA and γ-PGA SAP) at four different soil mass ratios (0.05% (1), 0.10% (2), 0.15% (3) and 0.20% (4)) to determine their effects on sandy loam soil hydro-physical properties. Both of them could reduce the cumulative infiltration of soil water. The total available water (TAW) which the soil water content (SWC) from field water capacity (FC) to permanent wilting point (PWP) after γ-PGA added into sandy loam soil had no significant different compared with CK, and the TAW was highest at the treatment of γ-PGA with 0.10% addition amount into sandy loam soil. However, the TAW of sandy loam soil increased dramatically with the γ-PGA SAP addition amount increasing. TM3 had the highest soil water absorption among the treatments with γ-PGA SAP. The T1 to T4 treatments with γ-PGA addition slightly prolonged retention time (RT) when SWC varied from FC to PWP compared with CK. For γ-PGA SAP addition treatments, the time for SWC varied from FC to PWP was 1.48 times (TM1), 1.88 times (TM2), 2.01 times (TM3) and 2.87 times (TM4) longer than that of CK, respectively. The results of this study will provide further information for the use of these materials in agricultural application.
| S-EPMC7983855 | biostudies-literature
Project description:Peptides (residues 1-42) (bovine prothrombin numbering) from bovine Factor X1 and X2 have been separately purified and digested before mass-spectrometric sequence assignment and identification of gamma-carboxyglutamic acid. N-terminal sequence was found to be identical, and 12 residues of gamma-carboxyglutamic acid were unambiguously identified. The new data give conclusive evidence for the N-terminal primary structure of bovine Factor X and extend present knowledge to show (i) unambiguous assignment of gamma-carboxyglutamic acid residues, including a previously unreported residue of gamma-carboxyglutamic acid at position 40 in both Factor X1 and X2, (ii) the physical difference between Factors X1 and X2 is not due to either different amino acid sequences or different gamma-carboxyglutamic acid contents of the N-terminal 42 residues.
| S-EPMC1186111 | biostudies-other
Project description:The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover ?-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.
| S-EPMC3724617 | biostudies-literature
Project description:The proline-rich gamma-carboxyglutamic acid (Gla) proteins (PRGPs) 1 and 2 are the founding members of a family of vitamin K-dependent single-pass integral membrane proteins characterized by an extracellular amino terminal domain of approximately 45 amino acids that is rich in Gla. The intracellular carboxyl terminal region of these two proteins contains one or two copies of the sequence PPXY, a motif present in a variety of proteins involved in such diverse cellular functions as signal transduction, cell cycle progression, and protein turnover. In this report, we describe the cloning of the cDNAs for two additional human transmembrane Gla proteins (TMG) of 20-24 kDa named TMG3 and TMG4. These two proteins possess extracellular Gla domains with 13 or 9 potential Gla residues, respectively, followed by membrane-spanning hydrophobic regions and cytoplasmic carboxyl terminal regions that contain PPXY motifs. This emerging family of integral membrane Gla proteins includes proline-rich Gla protein (PRGP) 1, PRGP2, TMG3, and TMG4, all of which are characterized by broad and variable distribution in both fetal and adult tissues. Members of this family can be grouped into two subclasses on the basis of their gene organization and amino acid sequence. These observations suggest novel physiological functions for vitamin K beyond its known role in the biosynthesis of proteins involved in blood coagulation and bone development. The identification and characterization of these proteins may allow a more complete understanding of the teratogenic consequences of exposure in utero to vitamin K antagonists, such as warfarin-based anticoagulants.
| S-EPMC29263 | biostudies-literature
Project description:L-γ-Carboxyglutamic acid (Gla) is an uncommon amino acid that binds avidly to mineral surfaces and metal ions. Herein, we report the synthesis of N-α-Fmoc-L-γ-carboxyglutamic acid γ,γ'-tert-butyl ester (Fmoc-Gla(O(t)Bu)(2)-OH), a suitably protected analogue for Fmoc-based solid-phase peptide synthesis. The residue was synthesized using a novel chiral Cu(II) complex, whose structure-based design was inspired by the blue copper protein rusticyanin. The five-coordinate complex is formed by Shiff base formation between glycine and the novel ligand (S)-2-(N-(2-methylthio)benzylprolyl)aminobenzophenone in the presence of copper. Michael addition of di-tert-butyl methylenemalonate to the α-carbon of the glycine portion of the complex occurs in a diastereoselective fashion. The resulting (S,S)-complex diastereomer can be easily purified by chromatography. Metal complex decomposition followed by Fmoc protection affords the enantiomerically pure amino acid. With the use of this novel chiral complex, the asymmetric synthesis of Fmoc-Gla(O(t)Bu)(2)-OH was completed in nine steps from thiosalicylic acid in 14.5% overall yield.
| S-EPMC3488861 | biostudies-literature
Project description:BackgroundMolasses is a wildly used feedstock for fermentation, but it also poses a severe wastewater-disposal problem worldwide. Recently, the wastewater from yeast molasses fermentation is being processed into fulvic acid (FA) powder as a fertilizer for crops, but it consequently induces a problem of soil acidification after being directly applied into soil. In this study, the low-cost FA powder was bioconverted into a value-added product of γ-PGA by a glutamate-independent producer of Bacillus velezensis GJ11.ResultsFA power could partially substitute the high-cost substrates such as sodium glutamate and citrate sodium for producing γ-PGA. With FA powder in the fermentation medium, the amount of sodium glutamate and citrate sodium used for producing γ-PGA were both decreased around one-third. Moreover, FA powder could completely substitute Mg2+, Mn2+, Ca2+, and Fe3+ in the fermentation medium for producing γ-PGA. In the optimized medium with FA powder, the γ-PGA was produced at 42.55 g/L with a productivity of 1.15 g/(L·h), while only 2.87 g/L was produced in the medium without FA powder. Hydrolyzed γ-PGA could trigger induced systemic resistance (ISR), e.g., H2O2 accumulation and callose deposition, against the pathogen's infection in plants. Further investigations found that the ISR triggered by γ-PGA hydrolysates was dependent on the ethylene (ET) signaling and nonexpressor of pathogenesis-related proteins 1 (NPR1).ConclusionsTo our knowledge, this is the first report to use the industry waste, FA powder, as a sustainable substrate for microbial synthesis of γ-PGA. This bioprocess can not only develop a new way to use FA powder as a cheap feedstock for producing γ-PGA, but also help to reduce pollution from the wastewater of yeast molasses fermentation.
| S-EPMC7594462 | biostudies-literature