Purification, amino acid sequence, and cDNA sequence of a novel calcium-precipitating proteolipid involved in calcification of corynebacterium matruchotii.
Corynebacterium matruchotii is a microbial inhabitant of the oral cavity associated with dental calculus formation. It produces membrane-associated proteolipid capable of inducing hydroxyapatite formation in vitro. This proteolipid was purified from chloroform:methanol extracts by chromatography on Sephadex LH-20 and migrated on SDS-polyacrylamide gel electrophoresis at 6-9 kDa. Removal of covalently attached acyl moieties by methanolic KOH decreased its molecular mass to approximately 5.5 kDa. The amino acid sequence of the apoproteolipid indicated a peptide of 50 amino acids, a calculated molecular weight of 5354 Da, and an isoelectric point of 4.28. Sequence analysis revealed an 8 amino acid sequence with homology to human phosphoprotein phosphatase 2A as well as several potential acylation sites and one phosphorylation site. The purified proteolipid induced calcium precipitation in vitro. Deacylation of the proteolipid by hydroxylamine treatment resulted in >50% loss of calcium-precipitating activity, suggesting that covalently attached lipids are required. Degenerate oligonucleotide primers, based on the amino acid sequence, were used to amplify the gene for the 5.5 kDa proteolipid from total chromosomal DNA of C. matruchotii by PCR. A 166 bp cDNA was isolated and sequenced, confirming the amino acid sequence of the proteolipid. Thus, we have sequenced a unique bacterial proteolipid that is involved in the formation of dental calculus by precipitating Ca2+ and possibly in transport of inorganic phosphate, necessary for hydroxyapatite formation.
van Dijk
S
S
Audie L. Murphy Memorial Veterans Affairs Medical Center, San Antonio, Texas 78229, USA.
Dean
D D
DD
Liu
Y
Y
Zhao
Y
Y
Chirgwin
J M
JM
Schwartz
Z
Z
Boyan
B D
BD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000032:analysis*; D001483:Base Sequence; D002113:Calcification, Physiologic / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D003352:Corynebacterium / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D008563:Membrane Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D018076:DNA, Complementary; D008563:Membrane Lipids; D011510:Proteolipids; D002118:Calcium