Investigation Of A Novel Metastasis Associated Gene Mag-1

Suppression subtractive hybridization (SSH) accompanied by DNA microarray is an effective method to isolate genes with different phenotype. Using this technique, 79 ESTs were found expressed two times or more in highly metastatic cell subpopulation PLA801 D than in poorly metastatic cell subpopulation PLA801 C. Both PLA801 C and PLA801 D were derived from the same parental lung giant cell carcinoma cell line PLA801 by single cell cloning technology, mag-1 encoding a polypeptide of 122 amino acids was obtained based on sequencing of the ESTs and sequence data mining.To further investigate the new metastasis associated gene, mag-1 was cloned into pET-28a vector and expressed in E. coli BL21 and the antibody of recombinant MAG-1 protein was prepared in rabbit. Using this antibody, intracelleular MAG-1 was analyzed by way of western blot. To our surprise, a protein band about 45KD was detected in PLA801 D.MAG-1 was found to share 100% identity with MGC11324 by blastP. The ORF of 1305bp encoding MGC11324 (434aa) was also found highly expressed in PLA801 D by RT-PCR. With specific probe, a transcript with 2.2kb was detected in human multiple tumor northern (MTN) blot, and an additional transcript of 3.5kb was only detected in the ovary tumor. Now it can be concluded that mag-1 and the gene encoding MGC 11324 is one gene.By bioimformatics analysis, the complete mag-1 is located at chromosome 4q21.23, and composed of 12 exons and 11 introns. Sequence analysis of MAG-1 protein shows that it contains a predicted signal peptide (amino acid 1-29) and an acyltransferase domain (amino acid 224-333); It is probably involved in metabolism. Prosite analysis reveals a cAMP and cGMP-dependent protein kinase phosphorylation site (amino acid 316-319), a leucine zipper (amino acid 156-177), six N-glycosylation sites(amino acid 228-231, 308-311, 309-312, 343-346, 422-425, 430-433), eight protein kinase C phosphorylation sites(amino acid 50-52, 57-59, 100-102, 113-115, 138-140, 185-187, 283-285, 344-346), five Casein kinasell phosphorylation sites(amino acid 57-60, 77-80, 117-120, 231-234, 373-376), and seven N-myristoylation sites(amino acid 3-8, 29-34, 223-228, 248-253, 252-257,304-309,405-410).