Identification and characterization of the carboxyl-terminal region of ratdentin sialoprotein

Two acidic proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (D PP), are present in the extracellular matrix of dentin but not in bone. The se two proteins are expressed in odontoblasts and preameloblasts as a singl e cDNA transcript coding a large precursor protein termed dentin sialophosp hoprotein (DSPP). DSPP is specifically cleaved into two unique proteins, DS P and DPP, However, the cleavage site(s) of DSPP and the mechanisms for reg ulating the cleavages are unknown. To identify the specific site(s) of DSPP that are cleaved when the initial translation product is converted to DSP and DPP, we performed a detailed analysis (Edman degradation and mass spect rometry) on selected tryptic peptides of a size originating from the COOH-t erminal region of rat DSP, After cleavage with trypsin, the DSP fragments w ere separated by a two-dimensional method (size-exclusion chromatography fo llowed by reversed phase high performance liquid chromatography). We charac terized 13 peptides from various regions of DSP, The analyses showed that p eptide Il(409)-Tyr(421) was the major COOH-terminal fragment, ending at Tyr (421) only 9 residues from the NH, terminus of DPP, Peptide Gln(385)-His(40 6) represented a second, minor COOH-terminal peptide that terminated at His (406), Both of these residues are well beyond the COOH terminus predicted p reviously by two independent studies estimating that rat DSP contained 360- 370 amino acids. Careful studies on two peptides showed that, among 9 poten tial casein kinase II phosphorylation sites, 2 serines were phosphorylated, We found that rat DSP was heterogeneous with respect to phosphorylation, b ecause this same peptide sequence eluted in two discrete peaks, one with 2 phosphoserines and the other having 1. The finding that 3 lysines just prec eding the COOH termini were modified by a 43-Da substituent (possibly a car bamoyl substituent) suggests that the lysines in this region were particula rly susceptible to attachment of this substituent.