Characterization of cementum derived growth factor as an insulin-like growth factor-I like molecule.
Cementum is the thin calcified outer layer through which tooth-root surfaces are anchored to soft periodontal connective tissues. A variety of growth factors and adhesion molecules are sequestered in the extracellular matrix of cementum, and we have purified and characterized one of the growth factors. This growth factor, the cementum derived growth factor (CGF), was purified from bovine cementum by acetic acid extraction followed by heparin affinity chromatography and HPLC using cation exchange, molecular sieve, and reverse-phase columns. NaDodSO4-polyacrylamide gel electrophoresis of purified CGF preparation revealed the presence of two major protein bands migrating with Mr 18,000-22,000 and 14,000-16,000. The latter was associated with the major part of the mitogenic activity. The activity of CGF was inhibited by antibodies to insulin-like growth factor-I (IGF-I) and IGF-I receptor. Both CGF and IGF-I were mitogenic to human gingival fibroblasts and alveolar bone cells, but the bone cells responded better to CGF than to IGF-I. The IGF-I did not bind to heparin-sepharose, while CGF bound to it and was eluted with 0.6M NaCl from heparin-sepharose columns. Heparin-sepharose 0.2M NaCl fractions of cementum extracts contained IGF-I migrating with Mr 7,500, but its mobility was not affected by N-glycosidase treatment. Western analysis using anti-IGF-I antibodies showed that CGF preparations contained cross-reacting species migrating with Mr 18,000-22,000, 14,000-16,000 and 11,000-12,000, however after treatment with N-glycosidase the Mr 18,000-22,000 component was absent. Internal amino acid sequences of six tryptic peptides of CGF were determined by microsequencing. The sequence of one 15-amino acid long peptide was the same as the receptor binding domain of IGF-I, and another 9-amino acid peptide had 78 % homology to a sequence derived from an untranslated region of sheep IGF-I exon 1. Four other peptides had no apparent homology with IGF-I. From these results we conclude that the CGF is an IGF-I like molecule.
Ikezawa
K
K
Department of Pathology, University of Washington, Seattle 98195, USA.
Hart
C E
CE
Williams
D C
DC
Narayanan
A S
AS
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D001842:Bone and Bones / Q000166:cytology; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003739:Dental Cementum / Q000737:chemistry*; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D005881:Gingiva / Q000166:cytology; D006133:Growth Substances / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017421:Sequence Analysis
D006133:Growth Substances; D007334:Insulin-Like Growth Factor I