Enamelysin mRNA displays a developmentally defined pattern of expression and encodes a protein which degrades amelogenin

Previously, a cDNA encoding a novel matrix metalloproteinase (enamelysin) was isolated from a porcine enamel organ-specific cDNA library. The cloned mRNA is tooth-specific and contains an open reading frame encoding a protein composed of 483 amino acids (Gene, 183:(1-2), p123-128, 1996). Here, we show that: 1) The expression of enamelysin mRNA is not limited to the enamel organ as previously reported. The enamelysin message is also expressed at very low levels in the pulp organ. 2) Northern analysis reveals that the enamelysin mRNA displays a developmentally defined pattern of expression in the enamel organ. The message is expressed at relatively high levels during the presecretory and early transition stages of development. However, during late maturation, the quantity of enamelysin mRNA is greatly reduced. Conversely, the low message levels in the pulp organ remain relatively constant throughout these developmental stages. 3) The enamelysin cDNA was ligated into a prokaryotic expression vector and recombinant enamelysin containing a His tag was purified from E. coli. Zymographic analysis utilizing recombinant murine amelogenin as the substrate, reveals that the purified enamelysin degrades amelogenin. Since enamelysin is developmentally regulated and is capable of degrading amelogenin, it is likely to play a significant role during enamel biomineralization.     

Enamelysin mRNA Displays a Developmental Defined Pattern of Expression and Encodes a Protein which Degrades Amelogenin

Previously, a cDNA encoding a novel matrix metalloproteinase (enamelysin) was isolated from a porcine enamel organ-specific cDNA library. The cloned mRNA is tooth-specific and contains an open reading frame encoding a protein composed of 483 amino acids (Gene, 183:(1–2), p123–128,1996). Here, we show that: 1) The expression of enamelysin mRNA is not limited to the enamel organ as previously reported. The enamelysin message is also expressed at very low levels in the pulp organ. 2) Northern analysis reveals that the enamelysin mRNA displays a developmentally defined pattern of expression in the enamel organ. The message is expressed at relatively high levels during the pre-secretory and early transition stages of development. However, during late maturation, the quantity of enamelysin mRNA is greatly reduced. Conversely, the low message levels in the pulp organ remain relatively constant throughout these developmental stages. 3) The enamelysin cDNA was ligated into a prokaryotic expression vector and recombinant enamelysin containing a His tag was purified from E. coli. Zymographic analysis utilizing recombinant murine amelogenin as the substrate, reveals that the purified enamelysin degrades amelogenin. Since enamelysin is developmentally regulated and is capable of degrading amelogenin, it is likely to play a significant role during enamel bio-mineralization.     

Degradation of enamel matrix proteins in porcine secretory enamel

To elucidate the progressive disappearance of 25 kDa amelogenin occurring in a narrow space near the surface of enamel, the alkaline soluble fraction which contained 80% of the total proteins was extracted from a newly formed porcine enamel. When this fraction was incubated with the addition of Ca ions in an in vitro system, the degradation of the coexisting amelogenin and enamelin occurred without activation during the incubation period. Although the fraction contained mainly two kinds of metalloproteinases, 56 kDa and 61 kDa gelatinolytic, and 41 kDa and 46 kDa caseinolytic activities, it was demonstrated on amelogenin enzymography that the caseinolytic one was concerned with the conversion of the 25 kDa amelogenin into the 20 kDa amelogenin. The protein distribution of the newly formed enamel indicated that the metalloproteinases degraded the coexisting enamelin and amelogenin imperfectly. Nevertheless, during the next developing stage they demonstrated their full activities. It is suspected that these activities are regulated by Ca ions, which may be increased by a cascade system.     

Expression of phosphoproteins and amelotin in teeth

The organic material of our teeth consists of collagens and a number of calcium-binding phosphoproteins. Six of these phosphoproteins have recently been grouped in the family of the SIBLINGs (small integrin-binding ligand, N-linked glycoproteins), namely osteopontin, bone sialoprotein, dentin matrix protein (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and enamelin. We prepared a cDNA library from rat incisors in order to identify the genes involved in tooth formation. The library was screened by subtractive hybridization with two probes; one specific for teeth, the other for bone. We found that the vast majority of the clones from our library were expressed at similar levels in bone and teeth, demonstrating the close relationship of the two tissues. Only 7% of all the clones were expressed in a tooth-specific fashion. These included clones for the enamel proteins; amelotin, amelogenin, ameloblastin and enamelin; for the dentin proteins DSPP and DMP1; and for the intermediate filament protein cytokeratin 13. Several typical bone proteins, including collagen I, osteocalcin, alkaline phosphatase and FATSO, were also expressed at significantly higher levels in teeth than in bone, probably due to the extreme growth rate of rat incisors. The amino acid sequence of rat amelotin showed 62% identity with the sequence from humans. It was expressed considerably later than the other enamel proteins, suggesting that amelotin may serve a function different from those of amelogenin, ameloblastin and enamelin.     

The Second Messenger Response upon Receptor Binding of Parathyroid Hormone in Rat Odontoblasts

To elucidate the progressive disappearance of 25 kDa amelogenin occurring in a narrow space near the surface of enamel, the alkaline soluble fraction which contained 80% of the total proteins was extracted from a newly formed porcine enamel. When this fraction was incubated with the addition of Ca ions in an in vitro system, the degradation of the coexisting amelogenin and enamelin occurred without activation during the incubation period. Although the fraction contained mainly two kinds of metalloproteinases, 56 kDa and 61 kDa gelatinolytic, and 41 kDa and 46 kDa caseinolytic activities, it was demonstrated on amelogenin enzymography that the caseinolytic one was concerned with the conversion of the 25 kDa amelogenin into the 20 kDa amelogenin. The protein distribution of the newly formed enamel indicated that the metalloproteinases degraded the coexisting enamelin and amelogenin imperfectly. Nevertheless, during the next developing stage they demonstrated their full activities. It is suspected that these activities are regulated by Ca ions, which may be increased by a cascade system.     

Amelogenin self-assembly and the role of the proline located within the carboxyl-teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.     

Amelogenin Self-Assembly and the Role of the Proline Located within the Carboxyl-Teleopeptide

A hallmark of biological systems is a reliance on protein assemblies to perform complex functions. We have focused attention on mammalian enamel formation because it relies on a self-assembling protein complex to direct mineral habit. The principle protein of enamel is amelogenin that self-assembles to form nanospheres. In mice, the principal amelogenin product is a 180 amino acid hydrophobic protein. The yeast two-hybrid assay has been used to demonstrate the importance of amelogenin self-assembly domains. We have generated specific variants of amelogenin to analyze contributions of individual amino acids to the self-assembly process. These amelogenin variants have been produced either by deleting carboxyl-terminal amino acids (to generate proteins that relate to the documented proteolytic products of mouse amelogenin) or by a site-directed mutagenesis approach. Assessment of variant amelogenins truncated at the carboxyl-terminal imply that the proline at position 169 of mouse amelogenin (M180) plays a significant role in amelogenin self-assembly. Site-directed mutagenesis of this particular proline, however, failed to disrupt the amelogenin self-assembly property. These conflicting data add to the complexity of protein-protein assembly mechanisms as they relate to the enamel matrix. Available data suggest a robustness of this enamel protein (amelogenin) that ensures a functional, even though mechanically less than optimal, enamel results despite either minor or major genetic errors to the amelogenin gene locus.     

Recent Advances in Amelogenin Biochemistry

This paper reviews advances in amelogenin biochemistry in three areas; (i) amelogenin expression; (ii) amelogenin post-translational and post-secretory processing, and (iii) amelogenin structure and function. Recent studies of amelogenin expression^1,2 have demonstrated that alternative-splicing of mouse amelogenin RNA generates seven distinct mRNAs, coding for amelogenin proteins from 194 to 44 amino acid residues in length. A polyclonal antibody to a sequence of the 194-residue murine amelogenin identified this protein in vivo. While several studies have reported that amelogenins are post-translationally phosphorylated, it has proved difficult to confirm this view. Mass spectrometry studies of bovine and porcine TRAP and LRAP amelogenins have established a phosphoserine residue at position-16 as originally reported by Takagi et al.³ for a 180-residue bovine amelogenin. Also, we discovered that the detailed mechanism(s) of carboxy-terminal amelogenin proteolytic processing appear different than previously reported.4 In terms of amelogenin structure, it is well known that amelogenins form aggregated structures. Studies employing a recombinant amelogenin and dynamic light-scattering instrumentation demonstrated aggregate structures of 15-20 nm in radius, corresponding to a mass of 2-3 million daltons. Imaging these aggregates by transmission electron and atomic force microscopy suggested that these structures are equivalent to the “stippled” or “granular” material seen in electron photomicrographs of developing enamel. Collectively, these advances in amelogenin biochemistry lead to a new view of amelogenin structure, processing and functions in enamel biomineralization.     

The cooperation of enamelin and amelogenin in controlling octacalcium phosphate crystal morphology

Enamel matrix proteins, including the most abundant amelogenin and lesser amounts of enamelin, ameloblastin, and proteinases, play vital roles in controlling crystal nucleation and growth during enamel formation. The cooperative action between amelogenin and the 32-kDa enamelin is critical to regulating the growth morphology of octacalcium phosphate crystals. Using biophysical methods, we investigated the interaction between the 32-kDa enamelin and recombinant pig amelogenin 148 (rP148) at pH 6.5 in phosphate-buffered saline (PBS). Dynamic light scattering results showed a trend of increasing particle size in the mixture with the addition of enamelin to amelogenin. Upon addition of the 32-kDa enamelin, the shift and intensity decrease in the ellipticity minima of rP148 in the circular dichroism spectra of rP148 illustrated a direct interaction between the 2 proteins. In the fluorescence spectra, the maximum emission of rP148 was blue shifted from 335 to 333 nm in the presence of enamelin as a result of complexation of the 2 proteins. Our results demonstrate that the 32-kDa enamelin has a close association with amelogenin at pH 6.5 in PBS buffer. Our present study provides novel insights into the possible cooperation between enamelin and amelogenin in macromolecular coassembly and in controlling enamel mineral formation.     

小鼠canstatin cDNA的克隆及其在大肠杆菌中的表达

目的:从小鼠肝脏组织克隆canstatin cDNA并在大肠杆菌(E.coli)中表达, 为进一步研究其抗肿瘤血管生成活性奠定基础。方法: 用Trizol试剂提取小鼠肝脏组织总RNA,通过RT-PCR扩增小鼠canstatin(m canstatin)的cDNA,克隆到pMD18-T载体中并进行序列分析。将小鼠canstatin cDNA 定向克隆于原核表达载体pET30a(+)中, 在大肠杆菌E.coli BL21中经IPTG诱导表达。结果:小鼠canstatin的cDNA长度为684bp,编码227个氨基酸,与已知的人canstatin的cDNA同源性为89%,氨基酸的同源性为96%。IPTG诱导原核表达载体pET30a(+)/m canstatin在大肠杆菌E.coli BL21中表达。结论: 首次成功克隆了小鼠canstatin的cDNA, 其原核表达载体pET30a(+)/m canstatin在大肠杆菌E.coli BL21中高效表达,小鼠canstatin抗肿瘤血管生成活性有待进一步研究。     

小鼠凋亡相关新基因TFAR15的克隆和序列分析

目的克隆人白血病细胞凋亡相关新基因TFAR15的小鼠同源序列 ,并比较其在不同种属间的序列同源性。方法利用EST(expressedsequencetag)拼排、RT PCR、DNA序列测定和计算机分析技术 ,对小鼠TFAR15进行研究。结果首次成功地进行了小鼠TFAR15全长cDNA的克隆和序列分析 ,发现小鼠TFAR15与小鼠其它cDNA没有明显的同源性 ,因此提交GenBank并被收录 ,登录号为AF159368。小鼠TFAR15和人TFAR15在核苷酸水平上有92.3 %的同源性 ,在氨基酸水平上有高达98.6 %的同源性 ;同时发现小鼠TFAR15在氨基酸水平上与线虫C14A4.11蛋白质有38 %的同源性。功能区分析发现 ,小鼠TFAR15cDNA序列含编码212个氨基酸的开放读码框架 ,有2个可能的N 糖基化位点 ,3个可能的caseinkinaseII磷酸化位点 ,4个可能的PKC磷酸化位点 ,并且可能是一种胞浆蛋白(cytoplasmicprotein)。结论小鼠TFAR15是进化上高度保守的新基因 ,可能具有重要的功能。