CLONING AND EXPRESSION OF CDNA-ENCODING HUMAN GALACTOCEREBROSIDASE, THE ENZYME-DEFICIENT IN GLOBOID-CELL LEUKODYSTROPHY

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galacto sylceramide, a major lipid in myelin, kidney and epithelial cells of s mall intestine and colon. We describe the molecular cloning of human G ALC cDNA and its expression in COS-1 cells. Degenerate PCR primers, de rived from N-terminal amino acid sequence from the 51 KDa band from hu man brain, were used to amplify cat testes RNA, and the resulting prod uct was used to screen human testes and brain libraries. Two overlappi ng clones contained the total protein coding region, while additional clones and PCR amplification were needed to obtain the complete 3' end of the cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007 bp of open reading frame (coding for 669 amino acids) , and 1741 bp of 3' untranslated sequence. Modification of the sequenc e surrounding the initiation codon to one more favorable for expressio n, resulted in a 6-fold increase in GALC activity in transfected COS-1 cells. The isolation of this clone will permit investigations into th e causes for GALC deficiency in humans and available animal models, de velopment of more accurate tests for patient and carrier identificatio n, and evaluation of methods for effectively treating GALC deficiency, initially using the animal models.