Synthesis of a potentially bioactive, hydroxyapatite-nucleating molecule.
A human phosphophoryn (PP) cDNA was previously cloned from immature root apex total RNA in our laboratory. This cDNA comprises 2,364 bp, encoding 788 amino acids. More than 80% of the sequences are arranged as (DSS)(n) (n = 1-16), DS, and NSS motifs. We hypothesize that the capability of PP to bind Ca(2+) and nucleate hydroxyapatite may depend on these repeated sequences. Two polypeptides were synthesized based on the human PP cDNA sequence to test the hypothesis. One polypeptide has the amino acid sequence DDPNSSDESNGNDD (synthetic polypeptide 1, SP1), which is from the N-terminal end of PP; the other polypeptide, DSKSDSSKSESDSS (synthetic polypeptide 2, SP2), is the PP repeated sequence motif. Phosphorylation of the polypeptides was accomplished by reacting them with adenosine triphosphate and casein kinases I and II. The ability of these molecules to cause mineralization was tested in a steady-state agarose gel system. The results show that phosphorylated SP2 (P-SP2) precipitated approximately 60% of the total Ca + PO(4) precipitated by PP. P-SP1 precipitated about 23% of that precipitated by PP and was similar to the amount precipitated in the control gel, that is, without added peptides. Transmission electron microscopy and X-ray diffraction analysis showed that the precipitate formed in the P-SP2-containing gel was hydroxyapatite. The capability of P-SP2 to nucleate Ca + PO(4) and precipitate hydroxyapatite is a result of the repeated sequence motif, which contains a high percentage of phosphorylated serine. This molecule could be used in the repair and regeneration of dental tissue.
Chang
S
S
Department of Cariology, Restorative Sciences, and Endodontics, University of Michigan, Ann Arbor, USA. renchang@umich.edu
Chen
H
H
Liu
J
J
Wood
D
D
Bentley
P
P
Clarkson
B
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D047389:Casein Kinase I / Q000378:metabolism; D047390:Casein Kinase II / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D005109:Extracellular Matrix / Q000737:chemistry; D006801:Humans; D006882:Hydroxyapatites / Q000737:chemistry*; D008854:Microscopy, Electron; D008963:Molar; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011498:Protein Precursors / Q000737:chemistry*; D014074:Tooth Calcification; D014961:X-Ray Diffraction
D018076:DNA, Complementary; D006882:Hydroxyapatites; D010455:Peptides; D010750:Phosphoproteins; D011498:Protein Precursors; C017630:phosphophoryn; D000255:Adenosine Triphosphate; D047389:Casein Kinase I; D047390:Casein Kinase II