Two types of HK deficiency have been reported, one in which activity is reduced exclusively in the RBC (HK r defect) and another with decreased activity in most tissues (HK l defect). HK r is induced in reticulocytes and decays dramatically during the maturation process to erythrocytes while HK l is constitutive and relatively stable. These findings suggested a separate genetic control of HK in the erythroid cells. We identified from poly(A)+ RNA of human reticulocytes a unique cDNA representing the RBC-specific hexokinase isozyme HK r. Its nucleotide sequence was identical to that of HK l cDNA, except for the 5' end. This cDNA lacked the porin-binding domain sequence; HK r does not bind to the mitochondria: this finding explains its cytoplasmic localization and lability. Instead of the porin-binding domain, the HK r cDNA included a unique sequence of 60 nucleotides at the beginning of the coding sequence, and a unique sequence upstream of the putative translation-initiation site. HK r was expressed in an erythroleukemic cell line (K562) and in reticulocytes, but not in a lymphocytic cell line. Using a probe derived from an HK r-specific sequence in the cDNA that included a unique upstream sequence, a clone was identified from a human genomic library. In the same clone, the first exon for Hk l was identified, including the porin-binding domain sequence. Thus, HK r and HK l appear produced by alternate usage of promoters and of splicing. The proximal promoter of HK l was extremely rich (≈80% content) in GC sequences generally found in housekeeping genes. On the other hand, the Hk r promoter was enriched with erythroid-specific sequences, including GATA, CACC, CCAAT and AP-1 elements. As found in other promoters of erythroid-specific genes, GATA elements appeared to substitute TATA elements. In addition, co-ordinated functions of GATA/GGAA/ATTA factors were suggested from EMSA analysis. HK r expression was inhibited whenγ-hemoglobin gene expression was induced in K562 cells by inducers of differentiation, such as DMSO, heme, and butyrate. The regulation of Hk r expression was further analyzed in K562 cells. These data confirm that HK r is a separate gene product expressed exclusively in the erythroid cells.
