Mass Spectrometry of Native Rat Amelogenins: Primary Transcripts, Secretory Isoforms, and C-terminal Degradation

Abstract

Cloning technologies have established unambiguously that amelogenins always seem larger in molecular weight (Mr) by gel electrophoresis (SDS-PAGE) than by mass spectrometry (MS). This has caused many problems relating cloned versions of amelogenin to proteins actually secreted by ameloblasts in vivo. In this study, discrete protein fractions at 31-20 kDa (Mr(SDS)) were prepared from freeze-dried rat incisor enamel by techniques optimized for preserving protein integrity. N-terminal sequence and amino acid compositional analyses indicated that the major protein forming these fractions was amelogenin. As expected, the molecular weights estimated by matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS were significantly less than their apparent molecular weights estimated by SDS-PAGE. Plots of Mr(SDS) vs. Mr(MS) for all fractions showed high linear correlation (r = 0.992). Analysis of MS data further indicated that the major protein in the 27-kDa fraction corresponded to the R180 secretory isoform of rat amelogenin, whereas some minor proteins in the 23-kDa fraction likely corresponded to a R156 secretory isoform. This was in contrast to major proteins forming the 25-, 24-, and 23-kDa fractions (Mr(SDS)), which seemed to represent proteolytic fragments of R180 progressively altered at the P169-A170, P164-L165, and F151-S152 C-terminal cleavage sites, respectively. Proteins in the 20-kDa fraction (Mr(SDS)) most closely matched by ESI-MS fragments of the R156 secretory isoform that were C-terminally-modified at the equivalent P164-L165 site.

Human osteoblastic cells discriminate between 20‐kDa amelogenin isoforms

“…Both A1 and A2 fractions had peaks at 16.6 kDa when measured by MALDI‐MS, a molecular mass that is consistent with the 20 kDa measured by SDS‐PAGE, and these results are supported by a study by C hen et al. (36). However, MALDI‐MS analyses indicate that the A1 fraction consists mainly of one molecular weight single charge peptide, whereas the A2 fraction contains multiple charged fractions of diverse molecular size, which are perhaps crucial for binding affinity, amelogenin structure, and function.…”

Mineral Acquisition Rates in Developing Enamel on Maxillary and Mandibular Incisors of Rats and Mice: Implications to Extracellular Acid Loading as Apatite Crystals Mature

“…(31,32) This value is very close to the known mass of the main R180 isoform of amelogenin present in rat incisor enamel (20,373 Da by mass spectrometry). (31) The amounts of acid released when creating stoichiometric hydroxyapatite with HPO 4 2− as precursor was computed from the following formula: (weight change per strip in micrograms/molecular mass of hydroxyapatite in nanomoles) × 8, assuming that all 8 mol of hydrogen ions are released directly for each mole of hydroxyapatite formed and the molecular mass of stoichiometric hydroxyapatite is 1004.64 [Ca 10 (PO 4 ) 6 (OH) 2 ]. (10,11) Correction factors for amounts of acid phosphate and carbonated apatite present in developing enamel, which lowered estimates of hydrogen ions released from 8.00 to 5.52-7.06 mol/ mol of mineral formed, were derived from speciation formulas reported by Aoba and Moreno (12) for pig enamel (none are available for rat enamel).…”

“…(11,29,30) The total weight of proteins in enamel strips was computed by subtracting the after-ashing weight from the starting dry weight of each strip. Because the molecular weights of amelogenins vary considerably in vivo (15,296 Da by MALDI mass spectrometry), (31) an average mass value between the heaviest and lightest component of the main amelogenin group was used in this calculation (19,032 Da). (31,32) This value is very close to the known mass of the main R180 isoform of amelogenin present in rat incisor enamel (20,373 Da by mass spectrometry).…”

“…Because the molecular weights of amelogenins vary considerably in vivo (15,296 Da by MALDI mass spectrometry), (31) an average mass value between the heaviest and lightest component of the main amelogenin group was used in this calculation (19,032 Da). (31,32) This value is very close to the known mass of the main R180 isoform of amelogenin present in rat incisor enamel (20,373 Da by mass spectrometry). (31) The amounts of acid released when creating stoichiometric hydroxyapatite with HPO 4 2− as precursor was computed from the following formula: (weight change per strip in micrograms/molecular mass of hydroxyapatite in nanomoles) × 8, assuming that all 8 mol of hydrogen ions are released directly for each mole of hydroxyapatite formed and the molecular mass of stoichiometric hydroxyapatite is 1004.64 [Ca 10 (PO 4 ) 6 (OH) 2 ].…”

Limited Proteolysis of Amelogenin: Toward Understanding the Proteolytic Processes in Enamel Extracellular Matrix

“…The activity of pig enamelysin was also examined under limited conditions on recombinant mouse amelogenin rM179, and the first cleavage site was found to be 151 F/S (Table 3). This cleavage site has also been detected in the in vivo fractions isolated from rat [31], in pig and rat developing tooth enamel [11], and on recombinant pig amelogenin in an in vitro system [9].…”