MMP-28, a new human matrix metalloproteinase with an unusual cysteine-switch sequence is widely expressed in tumors☆
Introduction
Remodeling of the extracellular matrix is an essential characteristic of a diversity of normal and pathological conditions. Complexity of cell-matrix interactions implicates a plethora of proteinases capable of cleaving the extracellular matrix. Matrix metalloproteinases (MMPs) are a comprehensive family of zinc metalloenzymes that are involved in the breakdown of extracellular matrix proteins (Massova et al., 1998). Identification of the expanding role of MMPs in complex regulatory and remodeling processes has stimulated the search for genes encoding for proteinases with a unique function, regulation and expression pattern.
Improved cloning strategies contributed to the recent progress in the search of novel MMPs (Leontovich et al., 2000, Llano et al., 1999, Pei, 1999a, Pei, 1999b, Pei, 1999c; Maidment et al., 1999, Velasco et al., 1999, Velasco et al., 2000, Wang et al., 1999). These strategies rely on the use of degenerate oligonucleotide primers in various polymerase chain reaction (PCR) protocols and large-scale sequencing. However, since there is an abundance of MMP genes with the known structure relative to the uncharacterized genes with much lower expression levels, the use of traditional cloning strategies may further have no or limited success. In our search for novel MMP genes, we combined subtraction of the known MMP genes from gene libraries with traditional PCR. This facilitated identification and cloning of three novel MMP genes with a relatively low abundance in human gene libraries. Here we report the molecular cloning, the nucleotide sequence, chromosomal location in the human genome, expression in human tissues and the genomic organization with a unique intron/exon distribution of one of our novel MMP cDNA gene. In accordance with the existing nomenclature, this novel MMP has been tentatively called MMP-28 (Nagase and Woessner, 1999).
Section snippets
Molecular cloning
Known MMP sequences were depleted from the first-strand mixed cDNA library representing MTC Human Panel I, Fetal Panel and Tumor Panel (Clontech, Palo Alto, CA) by using the following 19 biotinylated probes: U1 5′-AGGTGGACCAACAATTTCAGAGAG, U2 5′-GCAAGTGGGGCTTCTGCCCTGACC, U3 5′-CAATGGACAAAGGATACAACAGG, U4 5′-CTGGACGGATGGTAGCAGTCTAGG, U5 5′-TGGACCAACACCTCCGCAAATTAC, U6 (MMP-9) 5′-AGCGACAAGAAGTGGGGCTTCTGC, U7 5′-TGGACAGAAGATGCATCAGGCACC, U8 5′-ACCTGGACTATCGGGGATGACCAG, U9
Molecular cloning strategy
To facilitate the isolation of previously uncharacterized MMP genes we developed a novel cloning strategy. This strategy includes four major steps and combines the gene subtraction with PCR amplification employing the degenerate primers. The first step is a depletion of known MMP genes from a comprehensive multi-tissue first strand gene library. For these purposes we used biotin-labeled U1–U19 probes homologous to 19 known individual human MMPs (Fig. 1). The probes were allowed to hybridize the
Acknowledgements
Supported by grants from NIH CA83017 and CA77470, California BCRP 5JB0094 and Susan G. Komen Breast Cancer Foundation 9849 (to AYS)
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The nucleotide sequence reported in this paper has been submitted to the GenBank™ with Accession number AF315683.