Novel Full-Length cDNAs Differentially Expressed During Hematopoietic Commitment

Diabetes, Digestive, Kidney Diseases

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Abstract

We report the cloning of a novel myeloid lineage-restricted olfactomedin-related glycoprotein using mRNA differential display in conjunction with a modified two-phase liquid culture system. Early precursors of erythroid, myeloid, and mekagakaryotic lineages were enrich and isolated after induction with EPO, G-CSF, and TPO, respectively. RNA derived from the enriched cells was then subjected to differential display analysis to identify lineage-specific expressed genes. One clone, hGC-1,expressed specifically in early myeloid cells was further characterized. The cDNA clone of hGC-1 has a 2861 base pair sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510amino acids with a signal peptide and six motifs for N-linked glycosylation. The protein sequence of hGC-1 indicates that it is an olfactomedin-related glycoproteins, which include olfactomedin, TIGR, NOELIN-2 and latrophilin-1.Like other olfactomedin-like genes with tissue-restricted patterns ofexpression, hGC-1 is strongly expressed in prostate, small intestine, and colon, and moderately in bone marrow and stomach, and absent in other tissues. While hGC-1 is expressed during early normal myeloid differentiation, it is not expressed in K562, HL-60, MEG-01 or MOLT-4leukemia cell lines. The prepromyelocytic cell line HL-60 expressed hGC-1only when induced into granulocytic differentiation, but not with forced monocytic differentiation. Using a cell free in vitrotranscription/translation system, we have determined that translation ofhGC-1 yields a predominant protein band of approximately 54 kDa. Immunoprecipiation and immunolocalizition experiments using epitope-taggedhGC-1 demonstrate that the protein is a secreted extracellular protein. The hGC-1 gene locus has been mapped to chromosome 13q14.3. Together, our findings indicate that hGC-1 is primarily expressed as an extracellular olfactomedin-related glycoprotein during normal myeloid-specific lineage differentiation, suggesting the possibility of a matrix-related function forhGC-1 in differentiation and possibly tumor suppression.