Li Wu, Gao Cen, Yan Yan, DenBesten Pamela
Growth and Development Department, University of California at San Francisco, 521 Parnassus Ave., 94143-0640, USA.
Arch Oral Biol. 2003 Mar;48(3):177-83. doi: 10.1016/s0003-9969(02)00170-x.
Amelogenesis imperfecta (AI) is a group of inherited disorders with defective tooth enamel formation caused by various gene mutations. One of the mutations substitutes a cytidine for an adenine in exon 6 of the X-chromosomal amelogenin gene, which results in a proline to threonine change in the expressed amelogenin. This transformation is four amino acids N-terminal to the cleavage site for enamel matrix metalloproteinase-20 (MMP-20) in amelogenin. MMP-20 releases the tyrosine rich amelogenin peptide (TRAP) from amelogenin. This study evaluated the rate at which MMP-20 hydrolyses mutated amelogenin relative to unmutated amelogenin. A full-length recombinant human amelogenin and a mutated amelogenin with a substitution of proline by threonine were expressed and purified by ammonium sulphate precipitation and reverse phase HPLC. Recombinant metalloproteinase-20 (rMMP-20) was used to digest the recombinant proteins, which resulted in fragments with a mass predicted for TRAP. The proteolytic site was also modelled as substrates by two synthetic peptides, SYGYEPMGGWLHHQ and SYGYETMGGWLHHQ, selected from residues 36 to 49 of the amino acid sequence for amelogenin and the respective X-linked amelogenin mutant. These two peptides were labelled at their N- and C-termini respectively by using rhodamine and biotin. After digestion with MMP-20, the truncated peptides were separated by avidin-labelled magnetic Dynal beads and were identified by mass spectrometry. These results demonstrated that both oligopeptides were cleaved between tryptophan and leucine, matching the TRAP cutting site found in tooth enamel. Enzyme kinetics showed that the k(cat)/K(m) of rMMP-20 against the unmutated amelogenin peptide was 21 times greater than that against the mutated peptide. This study suggests that the reduced rate of TRAP formation by a single amino acid substitution alters enamel matrix hydrolysis by MMP-20, which may result in amelogenesis imperfecta.
釉质发育不全（AI）是一组由各种基因突变导致牙釉质形成缺陷的遗传性疾病。其中一种突变是在X染色体上的釉原蛋白基因外显子6中，将一个胞嘧啶替换为腺嘌呤，这导致所表达的釉原蛋白中脯氨酸变为苏氨酸。这种转变发生在釉原蛋白中釉基质金属蛋白酶-20（MMP-20）切割位点的N端四个氨基酸处。MMP-20从釉原蛋白中释放富含酪氨酸的釉原蛋白肽（TRAP）。本研究评估了MMP-20水解突变型釉原蛋白相对于未突变型釉原蛋白的速率。通过硫酸铵沉淀和反相高效液相色谱法表达并纯化了全长重组人釉原蛋白和脯氨酸被苏氨酸替代的突变型釉原蛋白。使用重组金属蛋白酶-20（rMMP-20）消化重组蛋白，产生了质量符合TRAP预测的片段。蛋白水解位点也通过两种合成肽SYGYEPMGGWLHHQ和SYGYETMGGWLHHQ作为底物进行建模，这两种肽分别选自釉原蛋白及其各自X连锁釉原蛋白突变体氨基酸序列的第36至49位残基。这两种肽分别在其N端和C端用罗丹明和生物素进行标记。用MMP-20消化后，截短的肽通过抗生物素蛋白标记的磁性Dynal珠进行分离，并通过质谱鉴定。这些结果表明，两种寡肽均在色氨酸和亮氨酸之间被切割，与在牙釉质中发现的TRAP切割位点相匹配。酶动力学表明，rMMP-20对未突变型釉原蛋白肽的k(cat)/K(m)比对突变型肽的大21倍。本研究表明，单个氨基酸替代导致TRAP形成速率降低，改变了MMP-20对釉基质的水解，这可能导致釉质发育不全。
