Enamel matrix protein turnover during amelogenesis: Basic biochemical properties of short-lived sulfated enamel proteins

Abstract

The formation and turnover of sulfated enamel proteins was investigated by SDS-PAGE, fluorography, and TCA-precipitations using freeze-dried incisors of rats injected intravenously with 35S-sulfate (35SO4) and processed at various intervals from 1.6 minutes to 4 hours thereafter. Some rats were injected first with 35SO4 followed 5 minutes later by 0.3 mg of cycloheximide. This was done to terminate protein translation and allow events related to extracellular processing and degradation of the sulfated enamel proteins to be visualized more distinctly. Other rats were injected with cycloheximide followed at 0 minutes (simultaneous injection) to 30 minutes later by 35SO4. This was done to characterize the time required for proteins to travel from endoplasmic reticulum to Golgi apparatus, where they became sulfated. The results indicated that enamel organ cells (ameloblasts) rapidly incorporated 35SO4 into a major approximately 65 kDa protein that was secreted into the enamel within 6-7.5 minutes. This parent protein appeared to be processed extracellularly within 15 minutes into major approximately 49 kDa and approximately 25 kDa fragments which themselves had apparent half-lives of about 1 and 2 hours, respectively. There were also many minor sulfated fragments varying in molecular weight (Mr) from approximately 13-42 kDa, which appeared to originate from extracellular processing and/or degradation of the parent approximately 65 kDa sulfated enamel protein or its major approximately 49 kDa and approximately 25 kDa fragments. Experiments with glycosidases further suggested that the majority of sulfate groups were attached to sugars N-linked by asparagine to the core of the approximately 65 kDa sulfated enamel protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis, Secretion, Degradation, and Fate of Ameloblastin During the Matrix Formation Stage of the Rat Incisor as Shown by Immunocytochemistry and Immunochemistry Using Region-specific Antibodies

“…These results raise the possibility that ameloblastin corresponds to the sulfated glycoprotein demonstrated by Smith et al (1995). However, experiments with glycosidases suggested that the majority of sulfate groups were attached to sugars that were connected to the protein via asparagine side-chains (N-linkages), resulting in the 65-kD sulfated glycoprotein (Smith et al 1995). Computer analysis of the ameloblastin cDNA sequence recognized no N-glycosylation target sites .…”

“…Matsuki et al (1995) constructed an unidirectional cDNA liberally from the growing end of the rat incisor, sequenced cDNA clones, classified their sequence, and showed that, among the enamel matrix-specific genes, the most redundant mRNAs next to amelogenin were for ameloblastin. These results raise the possibility that ameloblastin corresponds to the sulfated glycoprotein demonstrated by Smith et al (1995). However, experiments with glycosidases suggested that the majority of sulfate groups were attached to sugars that were connected to the protein via asparagine side-chains (N-linkages), resulting in the 65-kD sulfated glycoprotein (Smith et al 1995).…”

“…Using autoradiography of sections of rat incisor and fluorographs of proteins extracted from rat enamel organ and immature enamel after injection of various radioactive molecules, Smith and co-workers have revealed basic biochemical properties of short-lived sulfated glycoproteins (Smith et al 1995;Smith and Nanci 1996). The apparent molecular masses of some of these proteins (65,49,42,38,35, and 25 kD) were very similar to those of the post-translationally modified ameloblastin shown in this study (65, 48, 41, 36, 34, and 25 kD).…”

“…These proteins are believed to play significant roles during amelogenesis, such as the initiation of calcification (Robinson et al 1982;Aoba et al 1989;Diekwisch et al 1995) and the controlled growth of enamel crystals (Doi et al 1984;Aoba et al 1987;Fincham et al 1991). At present, several categories of enamel proteins, such as amelogenins (Eastoe 1979), enamelins (Termine et al 1980), tuftelin (Deutsch et al 1991), and sulfated glycoproteins (Fukae 1972;Miwa 1980;Sasaki et al 1982;Smith et al 1995;Smith and Nanci 1996), have been reported. We recently introduced a new category of enamel protein, which we termed sheath proteins (Uchida et al, 1995).…”

Tyrosyl Motif in Amelogenins BindsN-Acetyl-d-glucosamine

“…Dentine matrix contains both collagenous and non-collagenous proteins; the latter class also includes glycoproteins and phosphoproteins (6,(17)(18)(19)(20)(21). It has been shown that the sugar residues of the glycoconjugates are capable of inhibiting hydroxyapatite crystal formation (22)(23)(24).…”