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Transglutaminase Crosslinking of SIBLING Proteins in Teeth

¹ Division of Oral Biology, Faculty of Dentistry, McGill University, Strathcona Bldg.-Room M34, 3640 University Street, Montreal, QC, Canada H3A 2B2;
² Division of Experimental Medicine, Department of Medicine, Faculty of Medicine, McGill University, QC, Canada; and
³ Department of Anatomy and Cell Biology, Faculty of Medicine, McGill University, Montreal, QC, Canada;

View larger version (40K): [in this window] [in a new window]   Figure 1. Transglutaminase (TG) activity and protein substrates in tooth EDTA extracts. (A) Western blotting TG-activity assay; 5(biotin)pentylamine (bPA) labeling of tooth substrates by endogenous TG in tooth EDTA extract (E). bPA incorporation is detected by ExtrAvidin®-HRP conjugate and occurs in proteins having molecular weights of 60 and 80 kDa, and high-molecular-weight material above 150 kDa. The band at 100 kDa in the last lane represents TG2 itself, which autolabels in the crosslinking reaction. (B) Purification of bPA-labeled material by avidin chromatography and detection of BSP and DMP1 in the preparation by Western blotting with anti-BSP and anti-DMP1 antibodies; N-terminal sequencing gave the DPP sequence Asp-Asp-Pro-Asn. EAvid, ExtrAvidin®-HRP conjugate.

View larger version (31K): [in this window] [in a new window]   Figure 2. High-molecular-weight forms of SIBLING proteins in teeth and crosslinking by TG-Western blot analysis. (A) DPP polymers are present in the G2-extract. (B) DSPP, precursor protein for DPP and DSP, is found only in the E-extract. (C) Purified DPP forms polymers in vitro after TG2 incubation. (D) TG2 is found in HMW forms after incubation with DPP, indicating that it can serve as a crosslinking partner for DPP. (E) DMP1 polymers are found in both E- and G2-extracts. (F) Polymer formation of DMP1 after incubation with TG2, and bPA incorporation into DMP1 after TG2 incubation. (G) DSP is found only in monomeric forms in the E-extract. (H) DSP forms polymers and incorporates bPA when incubated with TG2. (I) BSP ‘dimers’ are found in both the E- and G2-extracts; similar-sized BSP forms are also found in rat and mouse bone E-extracts.

View larger version (81K): [in this window] [in a new window]   Figure 3. Immunohistochemical localization of SIBLING proteins in rat teeth. (A) TG2 and SIBLING proteins co-localize in predentin (TG2 and BSP) and along dentinal tubules (TG2, DPP, and DMP1), as represented by red/pink immunoreactivity over these tissue structures. DPP shows additional immunostaining of the intertubular dentin. (B) Immunostaining of cementocytes and their pericellular matrix in cellular cementum of molars. Od, odontoblast; PD, predentin; DEN, dentin; DT, dentinal tubules; Cc, cementocytes. Magnification bars equal 50 µm.

Journal of Dental Research, Vol. 84, No. 7, 607-612 (2005)
DOI: 10.1177/154405910508400705


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