Alvares K et al. (2009), Echinoderm phosphorylated matrix proteins UTMP1...

ECB-ART-41212

J Biol Chem 2009 Sep 18;28438:26149-60. doi: 10.1074/jbc.M109.024018.

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Echinoderm phosphorylated matrix proteins UTMP16 and UTMP19 have different functions in sea urchin tooth mineralization.

Alvares K , Dixit SN , Lux E , Veis A .

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Studies of mineralization of embryonic spicules and of the sea urchin genome have identified several putative mineralization-related proteins. These predicted proteins have not been isolated or confirmed in mature mineralized tissues. Mature Lytechinus variegatus teeth were demineralized with 0.6 N HCl after prior removal of non-mineralized constituents with 4.0 M guanidinium HCl. The HCl-extracted proteins were fractionated on ceramic hydroxyapatite and separated into bound and unbound pools. Gel electrophoresis compared the protein distributions. The differentially present bands were purified and digested with trypsin, and the tryptic peptides were separated by high pressure liquid chromatography. NH2-terminal sequences were determined by Edman degradation and compared with the genomic sequence bank data. Two of the putative mineralization-related proteins were found. Their complete amino acid sequences were cloned from our L. variegatus cDNA library. Apatite-binding UTMP16 was found to be present in two isoforms; both isoforms had a signal sequence, a Ser-Asp-rich extracellular matrix domain, and a transmembrane and cytosolic insertion sequence. UTMP19, although rich in Glu and Thr did not bind to apatite. It had neither signal peptide nor transmembrane domain but did have typical nuclear localization and nuclear exit signal sequences. Both proteins were phosphorylated and good substrates for phosphatase. Immunolocalization studies with anti-UTMP16 show it to concentrate at the syncytial membranes in contact with the mineral. On the basis of our TOF-SIMS analyses of magnesium ion and Asp mapping of the mineral phase composition, we speculate that UTMP16 may be important in establishing the high magnesium columns that fuse the calcite plates together to enhance the mechanical strength of the mineralized tooth.

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Genes referenced: LOC100887844 LOC100893907 LOC115919910 LOC115922275 LOC577317 LOC586799 LOC593358 LOC594261 LOC762863 pkcl2 SpP19L taf12 thrb

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	FIGURE 1. A, cross-section of developing L. variegatus tooth at an early stage of keel formation. Glutaraldehyde fixed teeth were processed as described by Veis et al. (16). This section was stained with toluidine blue. Cell nuclei stain a deep blue, and the non-mineralized syncytial matrix is a pale blue. The mineral-filled plates are evident as a dull blue-gray. B, the nuclei are seen trapped within the heavily mineralized layer just above the nuclei-rich cellular layer, sometimes referred to as the â€œstone partâ€ of the tooth. the umbo is at the top center of the flange. The syncytial layers interweave as they grow from each edge. This is the least mineralized portion of the tooth.
	FIGURE 2. Gel electrophoresis of the mineral-protected proteins extracted from the previously GdnHCl-extracted mineralized tooth powder with 0.6 n HCl. Gels A and C are stained with Stains-All, and gels B and D are stained with Coomassie. Lanes A1 and B1, the standard proteins; lanes A2 and B2, the total HCl-extracted proteins. After absorption on hydroxyapatite and subsequent high phosphate ion elution, the HA-bound proteins are shown in lanes C2 and D2, stained with Stains-All and Coomassie, respectively. The bands marked with an asterisk were the most prominent, and the proteins recovered from the gels in these bands were subject to trypsin degradation, and certain peptides were then subject to NH2-terminal sequencing.

	FIGURE 4. The complete nucleotide and amino acid sequences of L. variegatus UTMP19. A, the complete coding region nucleotide sequence of UTMP19. B, the amino acid sequence of L. variegatus UTMP19 compared with the predicted sequences of the two S. purpuratus P19 long and short isoforms. The probability of phosphorylation of threonine (boldface type, red) and serine (boldface type, green) residues at a level of >0.5 is indicated. The underlined residues have phosphorylation probability scores of 0.8â€“0.94. Note the four highlighted pairs of consecutive glutamic acid residues (boldface type, purple) within the most NH2-terminal portion of the molecule. The dashed line indicates residue identity. There are many more species differences in the COOH-terminal half of the molecule than in the NH2-terminal portions.
	FIGURE 5. The complete deduced sequences of L. variegatus UTMP16L and UTMP16S. The underlined sequence was that obtained from the amino acid sequence of tryptic peptide 8 (Fig. 4B) and verified by the cloned nucleotide sequences. The signal sequence, central Asp-Ser rich domain, and the transmembrane and cytosolic domains are all indicated. The dashed lines denote the residues deleted in UTMP16S. The high Gly content of the UTMP16L sequence from 32 to 75 suggests that it is a relatively flexible region with no clearly defined structure.
	FIGURE 6. Phosphorylation sites and phosphorylation landscape for UTMP16L. The list at the left shows that 22 residues are potentially capable of acting as kinase substrates with a greater than 0.5 probability. The Ser residues at 85, 89, 95, and 96 have the highest phosphorylation probability scores with casein kinase II (CKII) (green K) even without the possibility that phosphorylation would further enhance the potential for phosphorylation of additional Ser or Thr. residues. Thus, this central region exposed at the exterior of the cell syncytial membranes, but anchored by the transmembrane region to the cell interior, may be important in directing mineral localization. Other potential kinases are protein kinase C (red), protein kinase A (red), and casein kinase I (CKI) (black K). Note that several of the Ser residues may be substrates for either casein kinase I or II.
	FIGURE 7. The effect of potato acid phosphatase dephosphorylation on the apatite-bound and unbound fractions of the HCl-extracted proteins, as determined by the Stains-All staining of SDS-gel electrophoresis patterns before and after phosphatase digestion. M, molecular mass markers. Lane 1, unbound, undigested; lane 2, unbound, digested; lanes 3 and 6, blank; lane 4, bound, undigested; lane 5, bound, digested; lane 7, phosphatase plus buffer blank; lane 8, rat DPP (similar phosphorylated sequence control), undigested. Lane 9, phosphophoryn, digested. The faint pink in lane 7 is the control appearance of the acid phosphatase. The enhanced pink bands at âˆ¼45 and 20 kDa suggest that some proteins of DPP may interact stably with components in the phosphatase. Nevertheless, these data show clearly that the majority of the proteins in the mineral-associated preparations had been phosphorylated in vivo, indicating that the NetPhos predictions of phosphorylation were substantiated.

	FIGURE 9. The specificity of anti-UTMP16. Gel electrophoresis of the total HCl-extracted protein. Immunoprecipitation of this preparation with anti-UTMP16 yielded the single UTMP16 band shown in lane 1. Lane 2, the expected metachromatically blue stained bands.
	FIGURE 10. Immunolocalization of UTMP16 within a tooth cross-section early in the tooth development, where the primary and secondary plate mineralization has begun, and the keel has begun to develop. A, a total tooth cross-section (section 1090, âˆ¼10 mm from the origin of the plumula). B, the boxed region of A shown at higher resolution. The mineralized primary plates, developing secondary plates, and cellular parts of the cell syncytia are labeled. The UTMP16 fluorescence diffusely labels the syncytia in the less mineralized or unmineralized regions but concentrates brightly at the cell membrane boundaries of the calcite plates. This localization suggests that the UTMP16 moves to the syncytial membrane when mineralization is initiated. 1, cell syncytia; 2, forming primary plates, membrane labeling; 3, primary plates, mineralized; 4, forming secondary plates, membrane labeling; 5, mineralized secondary plates.

References [+] :

Alvares, The proteome of the developing tooth of the sea urchin, Lytechinus variegatus: mortalin is a constituent of the developing cell syncytium. 2007, Pubmed, Echinobase