A Specific Colorimetric Staining Method for γ-Carboxyglutamic Acid-Containing Proteins in Polyacrylamide Gels

Kang, Jie; Gijsbers, Birgit L. M. G.; Vermeer, Cees

doi:10.1006/abio.1995.1023

Cited by 13 publications

(11 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A portion of this solution was dialyzed against 50 mM HCl, dried, and aliquots of the resulting protein precipitate were analyzed by SDS-PAGE. The protein profile was revealed by staining either with Coomassie Brilliant Blue (Bio-Rad), or 4-diazobenzenesulfonic acid (Gla-specific stain; 8.5 mM 4-diazobenzene sulfonic acid (Sigma), as described (18). The crude precipitate was further purified by reverse phase-high performance liquid chromatography (RP-HPLC) to obtain pure GRP.…”

Section: Methodsmentioning

confidence: 99%

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

Viegas

Simes

Laizé

et al. 2008

Journal of Biological Chemistry

101

149

View full text Add to dashboard Cite

We report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 ␥-carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative ؉ 16 positive ‫؍‬ 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a ␥-carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being ␥-carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment.

show abstract

Section: Methodsmentioning

confidence: 99%

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

Viegas

Simes

Laizé

et al. 2008

Journal of Biological Chemistry

101

149

View full text Add to dashboard Cite

show abstract

“…The entire dialyzed extract was freeze‐dried, and two identical samples (approximately 30 μg of total protein) from each extract were loaded onto two adjacent lanes and analyzed by SDS‐PAGE. After electrophoresis, the two identical lanes were separated by cutting the gel in two, and the protein profile in each half was revealed by staining either with Coomassie Brilliant blue or with a Gla‐specific color reaction (23) as described below.…”

Section: Methodsmentioning

confidence: 99%

“…Total protein (20–30 μg) was dissolved in SDS sample buffer containing reducing agent (NuPage, Invitrogen), applied to a 12% or 4–12% gradient polyacrylamide precast gel containing 0.1% SDS (NuPage; Invitrogen) and run at constant 140 V. The gels were stained either with 0.2% Commassie Brilliant blue R‐250 (C.I. 42660; Bio‐Rad, Richmond, CA, USA), 10% trichloroacetic acid, 10% 5‐sulfosalicylic acid, or a DBS‐staining solution specific for Gla‐containing proteins (8.5 mM 4‐diazobenzene sulfonic acid [DBS]; Sigma, Madrid, Spain; 6.4 mM NaNO 2 in 2 M acetate buffer, pH 4.6), as described (23) . Blotting onto nitrocellulose (Invitrogen) was performed for 1 h at 80 mA using a Bio‐Rad Mini Trans‐Blot Cell system (Bio‐Rad) and a Bis‐Tris transfer buffer (NuPage; Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish

Simes

Williamson

Ortiz-Delgado

et al. 2003

Journal of Bone and Mineral Research

View full text Add to dashboard Cite

Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, 1 separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calc...

show abstract

“…1). Via specific staining (2) or HPLC detection techniques (3), these so-called Gla-proteins can be identified as unique products of vitamin K action. The vitamin K-dependent step is a carboxylation reaction that selectively takes place at a number of well-defined glutamate residues in only a small number of proteins (1,4).…”

Section: Structure and Function Of Vitamin Kmentioning

confidence: 99%

Role of vitamin K and vitamin K-dependent proteins in vascular calcification

Schurgers¹,

Dissel²,

Spronk³

et al. 2001

Zeitschrift f�r Kardiologie

Self Cite

View full text Add to dashboard Cite

A Specific Colorimetric Staining Method for γ-Carboxyglutamic Acid-Containing Proteins in Polyacrylamide Gels

Cited by 13 publications

References 0 publications

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish

Role of vitamin K and vitamin K-dependent proteins in vascular calcification

Contact Info

Product

Resources

About