RESUMEN
Vitamin K-dependent carboxylation of glutamic acid (Glu) residues into γ-carboxyglutamic acid (Gla) is a post-translational modification essential for normal protein activity of, for example, proteins involved in the blood coagulation system. These proteins may contain as many as 12 sites for γ-carboxylation within a protein sequence of 45 amino acid residues. In the biopharmaceutical industry, powerful analytical techniques are required for identification and localization of modified sites. We here present comparatively easy and rapid methods for studies of Gla-containing proteins using recent technology. The performances of two mass spectrometric fragmentation techniques, collision-induced dissociation (CID) and electron transfer dissociation (ETD), were evaluated with respect to γ-carboxylated peptides, applying on-line LC-ion trap MS. ETD MS has so far not been reported for Gla-containing peptides and the applicability of CID for heavily γ-carboxylated proteins has not been evaluated. The anticoagulant protein, protein C, containing nine Gla-sites, was chosen as a model protein. After tryptic digestion, three peptides containing Gla-residues were detected by MS; a 1.2 kDa fragment containing two Gla-residues, a 4.5 kDa peptide containing seven residues and also the 5.6 kDa tryptic peptides containing all nine Gla-residues. Regarding the shortest peptide, both CID and ETD provided extensive peptide sequencing. For the larger peptides, fragmentation by CID resulted in loss of the 44 Da CO(2)-group, while little additional fragmentation of the peptide chain was observed. In contrast, ETD resulted in comprehensive fragmentation of the peptide backbone. The study demonstrates that the combination of both techniques would be beneficial and complementary for investigation of γ-carboxylated proteins and peptides.
Asunto(s)
Ácido 1-Carboxiglutámico/análisis , Péptidos/química , Procesamiento Proteico-Postraduccional , Espectrometría de Masas en Tándem/métodos , Acilación , Secuencia de Aminoácidos , Fraccionamiento Químico/métodos , Cromatografía Liquida , Humanos , Datos de Secuencia Molecular , Sistemas en Línea , Proteína C/química , Proteínas Recombinantes/química , TripsinaRESUMEN
We have studied the atomic level structure of mineralized articular cartilage with heteronuclear solid-state NMR, our aims being to identify the inorganic species present at the surfaces of the mineral crystals which may interact with the surrounding organic matrix and to determine which components of the organic matrix are most closely involved with the mineral crystals. One-dimensional (1)H and (31)P and two-dimensional (1)H-(31)P heteronuclear correlation NMR experiments show that the mineral component is very similar to that in bone with regard to its surface structure. (13)C{(31)P} rotational echo double resonance experiments identify the organic molecules at the mineral surface as glycosaminoglycans, which concurs with our recent finding in bone. There is also evidence of gamma-carboxyglutamic acid residues interacting with the mineral. However, other matrix components appear more distant from the mineral compared with bone. This may be due to a larger hydration layer on the mineral crystal surfaces in calcified cartilage.
Asunto(s)
Calcificación Fisiológica , Cartílago Articular/química , Ácido 1-Carboxiglutámico/análisis , Animales , Huesos/química , Isótopos de Carbono , Articulaciones del Carpo , Durapatita/análisis , Femenino , Glicosaminoglicanos/análisis , Caballos , Hidrógeno , Masculino , Minerales/análisis , Resonancia Magnética Nuclear Biomolecular , Fósforo , Difracción de Polvo , Difracción de Rayos XRESUMEN
The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.
Asunto(s)
Ácido 1-Carboxiglutámico/química , Conotoxinas/química , Ácido 1-Carboxiglutámico/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Conotoxinas/genética , Conotoxinas/metabolismo , Caracol Conus/química , Cisteína/química , Cisteína/genética , ADN Complementario/genética , Cinética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Especificidad por SustratoRESUMEN
Vitamin K-dependent gamma-glutamyl carboxylase catalyzes the conversion of glutamyl residues to gamma-carboxyglutamate. Its substrates include vertebrate proteins involved in blood coagulation, bone mineralization, and signal transduction and invertebrate ion channel blockers known as conotoxins. Substrate recognition involves a recognition element, the gamma-carboxylation recognition site, typically located within a cleavable propeptide preceding the targeted glutamyl residues. We have purified two novel gamma-carboxyglutamate-containing conotoxins, Gla-TxX and Gla-TxXI, from the venom of Conus textile. Their cDNA-deduced precursors have a signal peptide but no apparent propeptide. Instead, they contain a C-terminal extension that directs gamma-carboxylation but is not found on the mature conotoxin. A synthetic 13-residue "postpeptide" from the Gla-TxXI precursor reduced the K(m) for the reaction of the Conus gamma-carboxylase with peptide substrates, including FLEEL and conantokin-G, by up to 440-fold, regardless of whether it was positioned at the N- or C-terminal end of the mature toxin. Comparison of the postpeptides to propeptides from other conotoxins suggested some common elements, and amino acid substitutions of these residues perturbed gamma-carboxylation of the Gla-TxXI peptide. The demonstration of a functional and transferable C-terminal postpeptide in these conotoxins indicates the presence of the gamma-carboxylation recognition site within the postpeptide and defines a novel precursor structure for vitamin K-dependent polypeptides. It also provides the first formal evidence to prove that gamma-carboxylation occurs as a post-translational rather than a cotranslational process.
Asunto(s)
Ácido 1-Carboxiglutámico/metabolismo , Conotoxinas/química , Conotoxinas/metabolismo , Procesamiento Proteico-Postraduccional , Ácido 1-Carboxiglutámico/análisis , Ácido 1-Carboxiglutámico/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Conotoxinas/genética , Conotoxinas/aislamiento & purificación , ADN Complementario/genética , Datos de Secuencia Molecular , Moluscos/genética , Moluscos/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por SustratoRESUMEN
Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational gamma-carboxy-glutamic acid (Gla) and beta-hydroxy aspartic acid (beta-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel beta-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.
Asunto(s)
Factor X/farmacología , Mutación Puntual , Proteínas Recombinantes/farmacología , Ácido 1-Carboxiglutámico/análisis , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análisis , Secuencia de Bases , Sitios de Unión , Línea Celular , ADN Complementario/genética , Activación Enzimática , Factor X/química , Factor X/genética , Trastornos Hemorrágicos/sangre , Trastornos Hemorrágicos/genética , Humanos , Enlace de Hidrógeno , Riñón/embriología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Prolina , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Serina , TransfecciónRESUMEN
The content of gamma-carboxyglutamic acid (gla) was determined in rat femoral bone and kidney cortex in rachitic and vitamin D treated animals. It was demonstrated that the level of gla is decreased in vitamin D depleted animals both in kidney cortex and femoral bone. Supplementation of vitamin D deprived animals with this vitamin resulted in an increase in the gla concentration to almost normal levels. Also the incorporation of 14C NaHCO3 in renal cortex microsomes from rachitic animals was blocked. It is suggested that the absence of gla resulted from the direct action of vitamin D on mRNA for the Ca-binding protein.