Echinoderm Phosphorylated Matrix Proteins UTMP16 and UTMP19 Have Different Functions in Sea Urchin Tooth Mineralization

Abstract

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.

Sea urchin tooth mineralization: Calcite present early in the aboral plumula

“…It is reasonable to hypothesize that some portion of the syncytial membrane nucleates the calcite of the primary plates; this as-of-yet- unidentified structure is found in every syncytial space of the tooth, perhaps at many places within a single space, but nucleation only occurs at one place and produces a highly specific calcite crystal orientation. Alvares et al (2009) have presented evidence that the sea urchin tooth protein UTMP16, is a membrane-anchored protein probably presenting an aspartic acid - serine rich sequence (GGSDTSSDTGSDDDSIDDDGSSDDSS) on the external side of the urchin tooth syncytial space and may be responsible for the mineral nucleation, similar to the proposal that the Asprich 2 peptide sequence (DEADEADADEADADEADADNDAADETAADVGTEAEDVADDE) (Gotliv et al 2005) may nucleate calcite on surfaces (Metzler et al 2010). Although the present x-ray scattering data cannot address whether ACC is injected into this space from vacuoles and rapidly crystallizes (or persists in contact with the calcite) or whether calcite grows directly from a mother liquor, the data clearly demonstrate that crystalline calcium carbonate (calcite) is present and originates in earliest stages of urchin tooth mineralization.…”

Genome-wide analysis of the skeletogenic gene regulatory network of sea urchins

“…Several PMC-specific, type I TM proteins, including P16, P58A and P58B, play essential roles in biomineral deposition (Cheers and Ettensohn, 2005;Adomako-Ankomah and Ettensohn, 2011). The precise biochemical functions of the P16 and P58 proteins are unknown, although P16 is phosphorylated and binds to hydroxyapatite (Alvares et al, 2009). A PMC-specific, GPIanchored carbonic anhydrase is likely to be involved in biomineral remodeling (Livingston et al, 2006).…”

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

“…The high phosphoserine content of the 22-kDa fish phosphoprotein is reminiscent of phosphophoryn, a cleavage product of dentin sialophosphoprotein belonging to the same small integrin binding ligand N-linked glycoprotein family of OPN and BSP, in which phosphorylation is important for the deposition of calcium phosphate crystals and that the acidic carboxylate groups alone are not sufficient (46,58). The generation of dentin sialophosphoprotein null mice resulted in tooth defects similar to human dentinogenesis imperfecta III (59).…”

“…To date, the studies related to ECM phosphoproteins of mineralizing tissues have been carried out in mammalian and avian vertebrates (1-15) as well as invertebrate species such as sea urchin (46,47). However, it is of major interest to study components of bone ECM in other vertebrate species such as fish.…”