Structure and transcriptional regulation of human alpha-mannosidase IIx (alpha-mannosidase II isotype) gene

Golgi alpha-mannosidase II is a key enzyme of N-glycan processing. Its genetic defect is associated with HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test). We previously cloned cDNAs of human alpha-mannosidase II (alpha-MII) and its isotype, alpha-mannosidase IIx [alpha-MII(x); Misago, M., Liao, Y. F, Eto, S., Mattei, M. G., Moremen, K. W & Fukuda, M. N. (1995) Proc. Natl Acad. Sci. USA 92, 11 766-11 770]. Constitutive expressions of alpha-MII and alpha-MII(x) mRNA were shown in various human tissues. To investigate the transcriptional regulation of alpha-MII(x) gene, we characterized the cosmid clone of 40-kb that includes the 5'-flanking sequence. This clone contains at least eight exons which encode 396 amino acid residues of a total of 1139 amino acid residues of alpha-MII(x). primer-extension analysis revealed multiple transcription-initiation sites in the range from -70 to -58 relative to the translation-initiation site. No canonical TATA or CAAT boxes were observed, but a (G+C)-rich region was found in close proximity to the transcription-initiation site. To localize the transcriptional regulatory region of this gene, various regions of the 5' sequences were fused to the luciferase gene, and transient-expression assays were conducted in human melanoma G-361 cells. These studies indicated that sequence from -12 to +11 relative to the most distal 5'-transcription-initiation site was involved in the promoter function. Within this region, the sequence GGGCGT similar to the consensus sequence of the Sp1 binding site, is present at positions -12 to -7. Enhancer activities were found in the region upstream of this site, notably from -4300 to -252. Thus, the alpha-MII(x) promoter located in a CpG island is also regulated by upstream elements, indicating the complexity of alpha-MII(x) gene expression.