Biomedical subjects

A G Fincham

Publications and source records attributed to A G Fincham.

At least 55 records · Page 3Linked to original sources

PubMed1994

Isolation and characterization of a mouse amelogenin expressed in Escherichia coli.

A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

Amelogenin↗

PubMed1994

Mass-spectrographic analysis of a porcine amelogenin identifies a single phosphorylated locus.

The amelogenins of the extracellular matrix of developing dental enamel, comprise a family of tissue-specific proteins which are postulated to play a central role in the biomineralization of dental enamel [1]. The primary structures of amelogenins derived from cow, pig, human, mouse and rat have now been elucidated by the interpretation of cDNA sequences or by direct amino acid sequence determinations [2-6] demonstrating a high degree of sequence homology between species [1]. However, the nature of post-translational modification of these proteins is less clear. In particular, early reports of amelogenin phosphorylation [7-8] have proved to be difficult to confirm by direct chemical analyses [1]. Using mass spectrographic analysis, we recently [9], reported that the lower molecular weight (5-7 kDa) bovine and porcine amelogenin polypeptides (TRAP and LRAP) contained a single phospho-serine residue at position 16Ser and, since these polypeptides are derived by proteolytic processing from the higher molecular weight "parent" amelogenins (18-25 kDa), we concluded that these precursor molecules must also be phosphorylated, as has previously been suggested [10]. In contrast to these observations, an extensive amino acid sequencing study of porcine amelogenins has recently reported no evidence for such phosphorylation [1]. We now report that a new analysis of the major porcine ("20K") amelogenin provides positive evidence for porcine amelogenin phosphorylation.

Amelogenin↗

PubMed1994

Alternative splicing of the mouse amelogenin primary RNA transcript.

A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5' ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5' untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.

Alternative Splicing↗

PubMed1994

Specific cleavage of a recombinant murine amelogenin at the carboxy-terminal region by a proteinase fraction isolated from developing bovine tooth enamel.

A proteinase fraction of 48-70-kDa was isolated from developing bovine tooth enamel by size exclusion and reversed-phase high-pressure liquid chromatography (HPLC) techniques. Proteolytic activity in the HPLC fraction was visualized by enzymography using gelatin as substrate. A recombinant murine amelogenin (M179) composed of 179 amino acid residues (20 kDa) was used as a substrate to examine the specificity of the enzymes in the isolated fractions. Incubation of M179 with the proteinase fraction at 37 degrees C generated a major proteolytic product eluting at about 42% acetonitrile from the reversed-phase column. This product had an amino-terminal sequence Pro-Leu-Pro-Pro-His-Pro- in conformity with that of the M179 parent protein. These data indicated that the product resulted from the cleavage of the M179 recombinant protein in the carboxy-terminal region. Mass spectroscopic analysis of the product isolated by reversed-phase HPLC gave a molecular mass of 18.89 kDa. Given an intact amino-terminal sequence, this mass figure suggests that this product terminates at Pro168 of the M179 residue sequence. The presence of EDTA in proteolysis experiments when M179 was used as substrate inhibited production of the 18.89-kDa product. Antipain, aprotinin, leupeptin and 4,(amidinophenyl)methanesulphonyl fluoride, which are serine proteinase inhibitors, did not affect the proteolytic activity. In addition, replacement of Ca2+ with Zn2+, Mn2+ or Co2+ in the proteolysis buffer inhibited the enzymatic activity. It is concluded that the 'high molecular-weight' proteinase cleaving M179 at Pro168-Ala169 is a specific 'calcium-dependent metalloproteinase'.

Amelogenesis↗

PubMed1993

Amelogenin post-translational modifications: carboxy-terminal processing and the phosphorylation of bovine and porcine "TRAP" and "LRAP" amelogenins.

TRAP and LRAP amelogenin components were isolated by size-exclusion and reversed-phase HPLC from developing dental enamel. Porcine developing enamel contains TRAP and LRAP components analogous to those of bovine. Amino acid composition and mass spectrographic analyses established that, in both species, the carboxy-terminal sequences of the LRAP components are two residues longer than previously reported for bovine LRAP, and that both the TRAPs and LRAPs contained a single phosphorylated residue. These amelogenin polypeptides were the principal components of the enamel protein lower molecular weight fraction. The LRAP sequence data for both species suggests that the mechanism of amelogenin carboxy-terminal processing may differ significantly from that previously suggested.

Amelogenin↗

PubMed1992

Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity.

A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.

Alternative Splicing↗

PubMed1991

Human developing enamel proteins exhibit a sex-linked dimorphism.

The amelogenin protein of developing dental enamel is generally accepted to mediate the regulation of the form and size of the hydroxyapatite crystallites during enamel biomineralization (1). A genetic disorder of enamel development (amelogenesis imperfecta) has been linked to the amelogenin gene AMEL(2-3), and loci regulating enamel thickness and tooth size have been mapped to the human sex chromosomes (4). In the human genome there are two AMEL loci with one copy of the gene on each of the sex chromosomes (AMELX and AMELY), whereas in the mouse only an AMELX locus is present (5). It is presently unknown if human AMELY is transcriptionally active. These observations prompted us to examine specimens of human developing enamel for sexual dimorphism at the protein level. We report here, for the first time, a diagnosis of differences in human enamel proteins which permits the distinction of specimens according to the sex of the individual.

Ameloblasts↗

PubMed1991

Amelogenin post-secretory processing during biomineralization in the postnatal mouse molar tooth.

The primary structures, molecular genetics and biosynthesis of the amelogenin protein of the developing tooth are established, but knowledge of their subsequent post-secretory processing and its relation to enamel biomineralization is fragmentary. Preparations of tooth matrix proteins were isolated from molars (M1) of mice from birth to 15 days and analysed by SDS-PAGE and immunochemical methods. Amelogenin proteins, isolated and partially purified by HPLC, were characterized by amino acid analysis and SDS-PAGE. At birth a 26 kDa amelogenin was present that during subsequent developmental stages generated a series of 20-25 kDa amelogenins differing in apparent size by approximately 1 kDa. Amino acid analyses showed that all these amelogenins have amino-terminal TRAP sequences; analyses for both glycosylation and phosphorylation were negative. It is suggested that these post-secretory amelogenins are generated by a sequence of specific carboxy-terminal cleavages, and that the observed post-secretory processing of amelogenin is functionally linked to the structure of the enamel matrix and the control of crystallite development.

Age Factors↗

PubMed1990

Isolation and partial characterization of a human amelogenin from a single fetal dentition using HPLC techniques.

A strategy based on high pressure liquid chromatography (HPLC) techniques for the isolation or a principal amelogenin molecule from a single human dentition is described. A partial sequence (33 residues) for this 24 kDa amelogenin is presented and related to earlier studies of human 5 kDa tyrosine-rich amelogenin polypeptides (TRAPs). A failure to identify amino acid residue #25 (tryptophan in other amelogenins) suggests that this 24 kDa amelogenin is the progenitor of the human TRAP-2 molecule and provides further support for the possibility of several human amelogenin gene products, generated by splice-junction selection, from the single amelogenin gene in the same individual. Alternatively, multiple amelogenins may arise by expression of both the AMELX and AMELY loci.

Amelogenin↗

PubMed1989

Human and mouse cementum proteins immunologically related to enamel proteins.

SDS-polyacrylamide gel electrophoresis, immunoblot and amino acid composition analyses were applied to human and mouse acellular cementum proteins immunologically related to enamelins and amelogenins. In this analysis, anti-mouse amelogenin, anti-human enamelin and synthetic peptide (e.g., -LPPHPGHPGYIC-) antibodies were shown to cross-react with tooth crown-derived enamelin with a molecular mass of 72,000 Da (72 kDa), amelogenins (26 kDa), and also to four human cementum proteins (72, 58, 50 and 26 kDa) and two mouse cementum proteins (72 and 26 kDa). Each of the antibodies recognized tooth root-derived cementum polypeptides which share one or more epitopes with tooth crown-derived enamel proteins. The molecular mass and isoelectric points for crown-derived and root-derived enamel-related proteins were similar. Analysis of human and mouse cementum proteins revealed a characteristic amino acid composition enriched in glutamyl, serine, glycine, alanine, proline, valine and leucine residues; compared to the major enamel protein amelogenin, cementum proteins were low in proline, histidine and methionine. The human and mouse putative intermediate cementum proteins appear to represent a distinct class of enamel-related proteins. Moreover, these results support the hypothesis that epithelial root sheath epithelia express several cementum proteins immunologically related to canonical enamel proteins.

Amelogenin↗

PubMed1989

Human amelogenins: sequences of "TRAP" molecules.

The extracellular protein matrix of developing enamel includes a major class of proteins, the amelogenins, which are believed to be concerned in regulating enamel biomineralization. Previous studies have shown the amelogenins of the extracellular matrix to be a complex of proline-rich hydrophobic proteins which, it is suggested, arise through posttranslational and postsecretory processing of a primary ameloblast gene product. More recently, it has been shown that the human amelogenin gene is located on both the X and Y chromosomes raising the possibility that polymorphism at the level of the gene may also contribute to the observed complexity of these enamel matrix proteins. To investigate such possible amelogenin polymorphism in developing human dental enamel, individual fractionated by size-exclusion and reversed-phase high pressure liquid chromatography (HPLC). Two tyrosine-rich amelogenin polypeptides (TRAPs) of approximately 5 kDa in size were isolated from an individual human dentition and characterized by automated gas-phase sequencing. These polypeptides were found to be of 42 (TRAP-2) and 44 (TRAP-1) amino acid residues in length; TRAP-2 lacked a carboxy-terminal -Gly-Trp sequence as has previously been described for analogous bovine TRAP molecules. However, residue #25 of the human TRAP-2 sequence was refractory to sequencing, apparently differing from the Trp-25 identified in TRAP-1. These findings suggest (1) two forms of TRAP molecules, differing only by cleavage of a carboxy-terminal dipeptide, are a general feature of human and other mammalian enamel proteins, probably being derived by postsecretory cleavage from the primary extracellular amelogenin; and (2) in human developing enamel four forms of TRAPs may arise either from polymorphism at the level of the gene, or by posttranscriptional alternative splicing of amelogenin mRNAs, coupled with specific post-secretory proteolytic processing.

Amelogenin↗

PubMed1989

Amelogenesis in vitro: a model for studies of epithelial postsecretory processing during tissue-specific extracellular matrix biomineralization.

The extracellular matrix (ECM) of developing mammalian enamel comprises a complex of unusual epithelial-derived proteins, which appear to function in concert to initiate and propagate tissue-specific biomineralization. Following enamel protein synthesis by ameloblast cells within the enamel organ, the subsequent steps of posttranslational modification, secretion, postsecretory processing and eventual removal of these proteins from forming enamel are largely unknown. To address this issue we have designed studies to investigate the hypothesis that enamel proteins are removed from enamel and translocated into the vasculature as relatively high-molecular-weight components. We examined enamel proteins recovered from serumless medium during prolonged organ culture of mouse capstage mandibular first molars. By 21 days in vitro the tooth crown formed and dentine and enamel biomineralization were apparent. At 31 days, explants retained metabolic activity and the enamel matrix showed extensive transformation. Immunologically identified enamel proteins of 26-18 k Da were produced by cultured tooth organs, translocated from tooth explants to the culture medium, recovered from the medium and then compared to control enamel protein from in vivo preparations. Comparable postsecretory processing of the 26-k Da amelogenin protein was observed in vitro and in vivo. We speculate that the pathway reported in the present studies is comparable to the processing of the enamel protein polypeptides of the maturing enamel which occurs in vivo. The in vitro organ culture model described in this report provides an approach with which to investigate the molecular events associated with epithelial-derived postsecretory processing of ECM molecules associated with tissue-specific biomineralization.

Ameloblasts↗

PubMed1989

Hertwig's epithelial root sheath differentiation and initial cementum and bone formation during long-term organ culture of mouse mandibular first molars using serumless, chemically-defined medium.

Studies were designed to test the hypothesis that Hertwig's epithelial root sheath (HERS) synthesizes and secretes enamel-related proteins that participate in the process of acellular cementum formation. Our experimental strategy was to examine sequential root development of the mouse mandibular first molar in vivo and in long-term organ culture in vitro using serumless, chemically-defined medium. Using anti-amelogenin, anti-enamelin and anti-peptide antibodies, enamel-related antigens were localized within intermediate cementum during HERS differentiation and root formation in vivo. Cap stage molars maintained for periods of up to 31 days in organ culture expressed morphogenesis and cytodifferentiation as identified by tooth crown and initial root, cementum and bone formation. Metabolically-labeled HERS products were analyzed by immunodetection using enamel-related antibodies and one- and two-dimensional SDS gel electrophoresis. A 72 kDa and 26 kDa polypeptide were identified in forming mouse cementum. Both of these root putative cementum proteins yield similar (identical) amino acid compositions; however, both proteins differed from the compositions of either mouse crown enamelin or amelogenin proteins. This approach provides a new and novel in vitro model towards understanding HERS differentiation and functions related to root and bone formation. The data support the hypothesis that HERS cells synthesize polypeptides related to but also different from canonical crown enamel proteins.

Animals↗

PubMed1989

Of mice and men: anatomy of the amelogenin gene.

Mammalian enamel matrix is composed of two principal proteins, the enamelins and amelogenin. Recombinant complementary DNA (cDNA) molecules for the predominant mouse amelogenin have been identified, characterized by direct determination of the DNA sequence, and used as a specific hybridization probe. The spatial- and temporal-restricted pattern for amelogenin gene expression within developing mouse molars has been traced at the level of a single cell using in situ hybridization. The mouse genome has been shown to contain only one copy of the amelogenin (AMEL) gene which is not amplified or rearranged during ameloblast determination. In contrast, the human genome contains two copies of the AMEL gene, one residing on the X chromosome and one upon the Y chromosome. These observations, the availability of specific enamel gene probes coupled with the application of new techniques in molecular biology now afford unique opportunities for the analysis of the molecular basis of inherited defects of human enamel such as amelogenesis imperfecta. Recent advances towards obtaining a physical map and the complete nucleotide sequence for the human genome, as well as the documented developmental biology, defined genetics and transgenic capability of the mouse, suggest that mouse and man are the most relevant and potentially informative models for analysis of normal and abnormal enamel biomineralization.

Amelogenesis Imperfecta↗

PubMed1985

DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein.

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional 10-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific genes.

Amelogenin↗

PubMed1983

Amelogenins. Sequence homologies in enamel-matrix proteins from three mammalian species.

Partial amino acid sequences for selected amelogenin polypeptides isolated from the developing enamel of cow, pig and human foetuses are reported. It was found that there was an identity of sequence for the initial 28 residues of the polypeptides analysed, irrespective of their origin or size. A tyrosine-rich polypeptide was shown to be the N-terminal fragment of the principal higher-molecular-weight amelogenins, although a leucine-rich polypeptide of similar size was not identified in any other amelogenin structure. The findings demonstrate a striking degree of sequence conservation for the amelogenin proteins of the extracellular enamel matrix and support the concept of a discrete fragmentation of an initial 30 000 Da amelogenin molecule during the mineralization of the enamel.

Amelogenin↗

PubMed1983

Bovine high molecular weight amelogenin proteins.

The existence of high molecular weight amelogenins in the matrix of developing fetal bovine dental enamel was investigated. After ion-exchange and gel filtration chromatography, a 40,000 dalton amelogenin protein has been isolated. This species was homogeneous on both guanidine hydrochloride gel filtration and sodium dodecyl sulfate (SDS) gel electrophoresis, but heterogeneous on conventional gel electrophoresis. This high molecular weight amelogenin may be a precursor to the complex of smaller sized amelogenins characteristic of the secretory phase of enamel formation.

Amelogenin↗

PubMed1983

Molecular composition of the protein matrix of developing human dental enamel.

The protein matrix of fetal human dental enamel was isolated and fractionated by chromatographic and electrophoretic procedures. Nine principal protein fractions were isolated and characterized. Possible inter-relationships of these proteins and their comparison with data from other species were examined. Significant differences between human proteins and those described for cow or pig enamel were identified.

Amino Acids↗