Bone And Tooth Matrix Biochemistry And Metabolism

National Institute Of Dental & Craniofacial Research

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Abstract

The Matrix Biochemistry Section focuses its research on the functions of five major noncollagenous proteins found associated with the mineralized matrix of bones and teeth. These include: bone sialoprotein (BSP); osteopontin (OPN); dentin matrix protein 1 (DMP1); dentin sialophosphoprotein (DSPP); and matrix extracellular phosphoglycoprotein (MEPE). We have made a strong case for the genetic relatedness of these seemingly different proteins and there is increasing acceptance of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family concept. The genes encoding these proteins are all clustered in a tandem fashion within a short (400,000 basepair) region of human chromosome 4 and they all have similar exon properties separated by type-0 introns. Because the same genes are clustered together in all mammals studied, they are likely to be the result of gene duplications and subsequent divergence >180 million years ago. One of the conserved motifs among the different SIBLING members (and among animal species for any single protein) is the integrin-binding tripeptide, arginine-glycine-aspartate (RGD), used to bind the SIBLING to the cell surface. [unreadable] [unreadable] In recent years, we have shown that at least three members of the SIBLING family bind and activate different members of the secreted matrix metalloproteinase (MMP) family as well as complement Factor H and may modify their activities by bridging these otherwise soluble proteins to cell surfaces. This year in a continuing collaboration with Dr. Fedarko of Johns Hopkins University, the specific kinetics of BSP's ablility to overcome the inhibitory effects of natural inhibitors and drugs known to interfere with MMP-2's ability to digest its substrates were studied. BSP was shown to have the property to renew MMP-2's ability to digest its substrates in the presence of drugs developed to stop the protease from acting. This renewal of activity was observed using both highly purified substrates and in tissue culture experiments, such as the ability to develop new blood vessels. One interesting implication of this work is that drugs which were designed to interfere with cancer metastasis and/or new blood vessel formation by blocking MMP activities, should also be screened to function in the presence of SIBLINGs, proteins well documented to be synthesized by cancer cells.[unreadable] [unreadable] The Section has started studying the biochemistry and genetics of the largest member of the SIBLING family, dentin sialophosphoprotein (DSPP). This protein is cleaved into two fragments, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). DPP, possibly the most acid and hydrophilic protein made by humans, it the most abundant non-collagenous protein found in dentin. Indeed, all of the known dentin-specific genetic diseases in humans map to the SIBLING family cluster and often specifically to DSPP. Although several human families with dentinogenesis imperfecta (DGI) or the less severe dentin dysplasia (DD) have been reported to have mutations in the first 5% of the DSPP protein sequence, most families have not had their mutations properly described. By modifying several biochemical and microbiological techniques, we were recently able to uncover the mutations in several of these families with both DGI and DD. The majority of the mutations were in the technically challenging DPP domain that is comprised of 230 repeats of a nominal 9-basepair motif that encodes the tripeptide, serine-serine-aspartic acid. In each case, the patients had lost one or four basepairs causing the normally soluble protein to become completely insoluble. This insoluble form of DPP was hypothesized to interfere with the production of the normal dentin matrix and to cause the teeth to become easily broken and worn to the gum line. Using our newly published insights and techniques, other laboratories will now be able to uncover the mutations in their DGI and DD patients.[unreadable] [unreadable] While studying the DGI and DD patients, we had to also verify that people without DD or DGI did not have similar losses of one or four bases in their DSPP gene. We noticed that while they did not have these frameshift-causing one or four basepair loss events, the normal population did have many differences among them. We found that among 100 normal people there were more than 37 different alleles within the DPP portion of the gene resulting from various combinations of numerous single nucleotide polymorphisms (SNPs) as well as changes in the total number of the 9-basepair repeats. A detailed look at these normal variants and the various possible mechanisms that could cause them, led us to hypothesize that the majority of the SNPs were caused by unusually rapid C-to-T changes in DNA sequences due to the deamination of 5-methyl-cytosine groups on both CpG and CpApG motifs. While the methylation of CpNpG trinucleotides have been studied in plants and a few mammals, to our knowledge this represents the first conclusive documentation of the epigenetic modulation of this motif in humans. The change in the number of repeats was hypothesized to be due to slip replication errors during meiosis, a mechanism known to occur in tandem repeat sequences. We observed that the diversity of DSPP sequences among humans (a species that is generally accepted to have gone through one or more recent genetic bottlenecks and is therefore genetically narrow in scope) is unusual compared to nearly all other coding sequences. This high rate of change in the DPP region of DSPP over the last 100,000 years or so may make it a useful marker for historic settlement and migration events.[unreadable] [unreadable] The Matrix Biochemistry Section freely gives probes (antisera, cDNA, proteins etc.) to any laboratory in the world that makes a reasonable request. In FY08, we sent 250 probes to 100 laboratories (15% of which were in the dental field) around the world. Others in the Branch have sent these same probes during this time as well.