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Splicing Determines the Glycosylation State of Ameloblastin

¹ Department of Biologic and Materials Sciences, Dental Research Lab, 1210 Eisenhower Place, Ann Arbor, MI 48108, and
² Department of Orthodontics and Pediatric Dentistry, University of Michigan School of Dentistry, 1011 N. University, Ann Arbor, MI 48109-1078, USA; and
³ Department of Periodontics and Endodontics and
⁴ Department of Biochemistry, School of Dental Medicine, Tsurumi University, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan

View larger version (60K): [in this window] [in a new window]   Figure 1. Purification of N-terminal Ambn cleavage products from developing porcine molars. (A) Chromatogram showing absorbance at 280 nm of alkaline extract proteins eluting from an ion exchange column. The contents of each fraction are shown on 5–20% gradient SDS-PAGE. Fraction Q1a (boxed) was used in subsequent purification steps. (B, left) 18% Tris/glycine gel showing fractionation of Q1a by precipitation of amelogenins with TBS (pH 7.4). Western blot with Ambn-63 shows that ameloblastin N-terminal cleavage products are in the supernatant (Sup), but not the precipitate (Ppt). (B, right) 18% Tris/glycine gel and Western blots showing Q1a-Sup fractionation by PNA affinity binding: UnB, unbound; washes (W1, W2, W3), eluted (E), and PNA beads (B). (C) Chromatogram showing absorbance at 220 nm of fraction Q1a-Sup-Ub separated by NPS RP-HPLC and SDS-PAGE and Western blots of the 12 fractions. (D) Chromatogram showing absorbance at 220 nm of fraction Q1a-Sup-E separated by NPS RP-HPLC and SDS-PAGE and Western blots of the 4 fractions. Ambn-positive fractions (boxed) were selected for further analyses.

View larger version (71K): [in this window] [in a new window]   Figure 2. Lectin staining of final fractions containing N-terminal Ambn cleavage products. RP-HPLC fractions 2, 5, and 7 that did not bind to the PNA affinity beads (UnB) and RP-HPLC fractions 2 and 3 eluted (E) from the beads were separated by SDS-PAGE, transferred to membranes, and tested for their ability to bind Ambn-63 antibody and 7 different lectins with various carbohydrate specificities. The lectins and their binding specificities were: PNA (galactose), MPA (N-acetyl-galactosamine), LPA (sialic acid), Con A (mannose), UEA-I (fucose), GS-II (N-acetyl-glucosamine), and WGA (N-acetyl-glucosamine). These results suggest an O-linked glycosylation having a structure like that shown on the lower right. Protein bands selected for N-terminal sequencing are labeled in the SDS-PAGE on the upper left. Bands a through f all gave the Ambn N-terminal sequence VPAFPRQP... . Bands marked with an asterisk gave the amelogenin N-terminal sequence MPLPPHPG... . This confirmed that the lectin-positive bands were ameloblastin N-terminal cleavage products. Based upon integration of the chromatographic peaks (2 and 3 for the PNA-bound and 5 for the PNA-unbound), the abundance of Ambn395 relative to Ambn380 is at least 2:1. We say a minimum of 2:1, because it is apparent that the PNS-bound material is almost all Ambn, while the unbound fractions still contain some amelogenin.

View larger version (34K): [in this window] [in a new window]   Figure 3. (left) Chromatogram showing absorbance at 230 nm of Pronase-digested Ambn N-terminal cleavage products separated on a Sephadex G-25 column. The contents of peak 3 were positive for glycosylation with the phenol-sulfuric acid colorimetric assay, and gave the Edman sequence: HPPPLPXQPS. The blank cycle for Ser86 identifies this as the position of the O-linked glycosylation. (right) Deduced amino acid sequence of the 395-amino-acid ameloblastin. Protein sequences characterized in this study are boxed. Underlined amino acid positions are modified following translation. The 15-amino-acid sequence deleted by alternative splicing is in bold. Previously determined ameloblastin cleavage sites from in vivo sources are marked by arrowheads (Iwata et al., 2007).

Journal of Dental Research, Vol. 86, No. 10, 962-967 (2007)
DOI: 10.1177/154405910708601009


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