Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ

doi:10.1016/S0378-1119(96)00525-2

Gene

Volume 183, Issues 1–2, 1996, Pages 123-128

https://doi.org/10.1016/S0378-1119(96)00525-2 Get rights and content

Abstract

A cDNA encoding a novel matrix metalloproteinase (MMP) was isolated from a porcine enamel organ-specific cDNA library. Multiple tissue northern blot analysis revealed the presence of two mRNA transcripts which were expressed only in the enamel organ. The transcripts were 1968 bp or 3420 bp in length and resulted from the utilization of alternative polyadenylation sites. The open reading frame of the cloned mRNA encodes a protein composed of 483 amino acids. The MMP has a predicted molecular mass of 54.1 kDa, which is similar to that of the stromelysins or collagenases, although it is not a member of either of these two classes of MMPs. A motif analysis revealed that the cloned MMP does not contain a consensus hemopexin-like domain because it lacks a critical tryptophan and proline residue at the appropriate positions. Since the cloned MMP is a new member of the MMP gene family and its expression appears limited to the enamel organ, we have named it enamelysin.

References (28)

P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction
Anal. Biochem.
(1987)
I.E. Collier et al.
H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen
J. Biol. Chem.
(1988)
P.K. Denbesten et al.
Separation by polyacrylamide gel electrophoresis of multiple proteases in rat and bovine enamel
Arch. Oral. Biol.
(1989)
J.M. Freije et al.
Molecular cloning and expression of collagenase-3, a novel human matrix metalloproteinase produced by breast carcinomas
J. Biol. Chem.
(1994)
G.I. Goldberg et al.
Human fibroblast collagenase
K.A. Hasty et al.
Human neutrophil collagenase
C. Heussen et al.
Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates
Anal. Biochem.
(1980)
D.E. Kleiner et al.
Quantitative zymography: detection of picogram quantities of gelatinases
Anal. Biochem.
(1994)
S.D. Shapiro et al.
Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase
J. Biol. Chem.
(1992)
S.D. Shapiro et al.
Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages
J. Biol. Chem.
(1993)

T. Takino et al.

Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library

S.M. Wilhelm et al.

SV40-transformed human lung fibroblasts secrete a 92-kDa type IV collagenase which is identical to that secreted by normal human macrophages

J. Biol. Chem.

(1989)

R.T. Aimes et al.

Cloning of a 72 kDa matrix metalloproteinase (gelatinase) from chicken embryo fibroblasts using gene family PCR: expression of the gelatinase increases upon malignant transformation

Biochem. J.

(1994)

P. Basset et al.

A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas

Nature

(1990)

Cited by (176)

Trafficking and secretion of keratin 75 by ameloblasts in vivo
2019, Journal of Biological Chemistry
A highly specialized cytoskeletal protein, keratin 75 (K75), expressed primarily in hair follicles, nail beds, and lingual papillae, was recently discovered in dental enamel, the most highly mineralized hard tissue in the human body. Among many questions this discovery poses, the fundamental question regarding the trafficking and secretion of this protein, which lacks a signal peptide, is of an utmost importance. Here, we present evidence that K75 is expressed during the secretory stage of enamel formation and is present in the forming enamel matrix. We further show that K75 is secreted together with major enamel matrix proteins amelogenin and ameloblastin, and it was detected in Golgi and the endoplasmic reticulum (ER)–Golgi intermediate compartment (ERGIC) but not in rough ER (rER). Inhibition of ER–Golgi transport by brefeldin A did not affect the association of K75 with Golgi, whereas ameloblastin accumulated in rER, and its transport from rER into Golgi was disrupted. Together, these results indicate that K75, a cytosolic protein lacking a signal sequence, is secreted into the forming enamel matrix utilizing portions of the conventional ER–Golgi secretory pathway. To the best of our knowledge, this is the first study providing insights into mechanisms of keratin secretion.
Porcine keratin 75 in developing enamel
2019, Journal of Oral Biosciences
To provide in vivo biochemical evidence for the isolation, identification, and characterization of porcine keratin 75 (K75) in developing enamel.
Immunolocalization of K75 was observed in mandibles from mice at postnatal days 5 and 11. K75 gene expression was analyzed by quantitative reverse transcription-polymerase chain reaction using enamel organ epithelium (EOE) of incisors from pigs at 5 months of age. Enamel protein was extracted and isolated from both immature and mature enamel of second molars from 5-month-old pigs, and the K75 antibody-positive fraction was analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). In vitro protease digestion of K75-antibody-positive fraction was carried out using porcine kallikrein 4 (pKLK4) or recombinant human enamelysin (rhMMP20) and their degradation patterns were characterized by both SDS-PAGE and western blotting.
Specific immunostaining for K75 was restricted to the layers of stratum intermedium and the enamel side of ameloblasts in mice at postnatal day 5, and to the papillary layer at postnatal day 11. Porcine K75 was expressed throughout enamel formation, but its transcript levels were significantly higher in the transition EOE than in the secretory- and maturation-stage EOE. Porcine K75 was extracted from the neutral soluble fraction from both immature and mature enamel. It was identified by LC-MS/MS analysis, and was found not to be degraded by either pKLK4 or rhMMP20.
We propose that K75 is present in the developing enamel and undergoes different processing/degradation compared to other enamel proteins.
Deviations of inorganic and organic carbon content in hypomineralised enamel
2015, Journal of Dentistry
The purpose of this study was to discriminate hypomineralised enamel of permanent first molars from normal enamel by means of spectroscopic methods.
The present study was conducted using Multi spot Raman Fourier Transform Spectroscopy, Diffuse Reflectance Infrared Fourier Transform Spectroscopy (FTIR) and X-ray diffraction (XRD).
Raman spectroscopy indicated significantly more B-type carbonate and hydrocarbons in hypomineralised enamel diagnosed as MIH (Molar Incisor Hypomineralisation). From XRD analysis, no changes in crystallinity of the enamel apatite could be found.
Using multi spot Raman-spectroscopy, a significant molecular discrimination between normal and hypomineralised enamel could be made.
Detailed surface studies are needed in order to achieve better restorative materials, specifically designed for restoration of hypomineralised enamel, and are also needed in order to understand and predict the clinical consequences of hypomineralised enamel with the condition MIH.
Intrinsically disordered enamel matrix protein ameloblastin forms ribbon-like supramolecular structures via an N-terminal segment encoded by exon 5
2013, Journal of Biological Chemistry
Tooth enamel, the hardest tissue in the body, is formed by the evolutionarily highly conserved biomineralization process that is controlled by extracellular matrix proteins. The intrinsically disordered matrix protein ameloblastin (AMBN) is the most abundant nonamelogenin protein of the developing enamel and a key element for correct enamel formation. AMBN was suggested to be a cell adhesion molecule that regulates proliferation and differentiation of ameloblasts. Nevertheless, detailed structural and functional studies on AMBN have been substantially limited by the paucity of the purified nondegraded protein. With this study, we have developed a procedure for production of a highly purified form of recombinant human AMBN in quantities that allowed its structural characterization. Using size exclusion chromatography, analytical ultracentrifugation, transmission electron, and atomic force microscopy techniques, we show that AMBN self-associates into ribbon-like supramolecular structures with average widths and thicknesses of 18 and 0.34 nm, respectively. The AMBN ribbons exhibited lengths ranging from tens to hundreds of nm. Deletion analysis and NMR spectroscopy revealed that an N-terminal segment encoded by exon 5 comprises two short independently structured regions and plays a key role in self-assembly of AMBN.
Background: Ameloblastin plays a key role in the complex biomineralization process that forms tooth enamel, the hardest tissue of the body.
Results: Ameloblastin self-associates into ribbon-like supramolecular structures via a short segment encoded by exon 5.
Conclusion: Ameloblastin self-association may be essential for correct structural organization and mineralization of the enamel in vivo.
Significance: The results provide molecular insight into biology of tooth enamel formation.
The proteolytic processing of amelogenin by enamel matrix metalloproteinase (MMP-20) is controlled by mineral ions
2013, Biochimica et Biophysica Acta - General Subjects
Enamel synthesis is a highly dynamic process characterized by simultaneity of matrix secretion, assembly and processing during apatite mineralization. MMP-20 is the first protease to hydrolyze amelogenin, resulting in specific cleavage products that self-assemble into nanostructures at specific mineral compositions and pH. In this investigation, enzyme kinetics of MMP-20 proteolysis of recombinant full-length human amelogenin (rH174) under different mineral compositions is elucidated.
Recombinant amelogenin was cleaved by MMP-20 under various physicochemical conditions and the products were analyzed by SDS-PAGE and MALDI-TOF MS.
It was observed that mineral ions largely affect cleavage pattern, and enzyme kinetics of rH174 hydrolysis. Out of the five selected mineral ion compositions, MMP-20 was most efficient at high calcium concentration, whereas it was slowest at high phosphate, and at high calcium and phosphate concentrations. In most of the compositions, N- and C-termini were cleaved rapidly at several places but the central region of amelogenin was protected up to some extent in solutions with high calcium and phosphate contents.
These in vitro studies showed that the chemistry of the protein solutions can significantly alter the processing of amelogenin by MMP-20, which may have significant effects in vivo matrix assembly and subsequent calcium phosphate mineralization.
This study elaborates the possibilities of the processing of the organic matrix into mineralized tissue during enamel development.
MMP20 and KLK4 activation and inactivation interactions in vitro
2013, Archives of Oral Biology
Enamelysin (MMP20) and kallikrein 4 (KLK4) are believed to be necessary to clear proteins from the enamel matrix of developing teeth. MMP20 is expressed by secretory stage ameloblasts, while KLK4 is expressed from the transition stage throughout the maturation stage. The aim of this study is to investigate the activation of KLK4 by MMP20 and the inactivation of MMP20 by KLK4.
Native pig MMP20 (pMMP20) and KLK4 (pKLK4) were isolated directly from enamel scrapings from developing molars. Recombinant human proKLK4 (rh-proKLK4) was activated by incubation with pMMP20 or recombinant human MMP20 (rhMMP20), and the resulting KLK4 activity was detected by zymography. Reaction products were isolated by reverse-phase high performance liquid chromatography (RP-HPLC), and their N-termini characterized by Edman degradation. The pMMP20 was incubated with pKLK4 under mildly acidic or under physiologic conditions, and enzyme activity was analyzed by zymography. The catalytic domain of rhMMP20 was incubated with pKLK4 or recombinant human KLK4 (rhKLK4) and the digestion products were characterized by zymography and Edman degradation.
Both pMMP20 and rhMMP20 activated rh-proKLK4 by cleaving at the propeptide-enzyme junction used in vivo. The pMMP20 was inactivated by pKLK4 under physiologic conditions, but not under mildly acidic conditions. Both pKLK4 and rhKLK4 cleaved MMP20 principally at two sites in the catalytic domain of MMP20.
MMP20 activates proKLK4 and KLK4 inactivates MMP20 in vitro, and these actions are likely to occur during enamel formation in vivo.

View all citing articles on Scopus

View full text

Molecular cloning and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ

Abstract

Anal. Biochem.

J. Biol. Chem.

Arch. Oral. Biol.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cloning of a 72 kDa matrix metalloproteinase (gelatinase) from chicken embryo fibroblasts using gene family PCR: expression of the gelatinase increases upon malignant transformation

Biochem. J.

A novel metalloproteinase gene specifically expressed in stromal cells of breast carcinomas

Nature