CLONING AND CHARACTERIZATION OF CDNA-ENCODING CANINE ALPHA-L-IDURONIDASE - MESSENGER-RNA DEFICIENCY IN MUCOPOLYSACCHARIDOSIS-I DOG

Alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which cau ses mucopolysaccharidosis I (MPS I); a canine MPS I colony has been br ed to test therapeutic intervention. The enzyme was purified to appare nt homogeneity from canine testis and found to consist of two electrop horetically separable proteins that had common internal peptides but d iffered at their amino termini. A 57-base oligonucleotide, correspondi ng to the most probable codons of the longest peptide, was used to scr een a canine testis cDNA library. Three cDNAs were isolated, two of wh ich lacked the 5'-end whereas the third was full-length except for a s mall internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The am ino terminus of the larger protein, glutamic acid 26, is at the predic ted signal peptide cleavage site, whereas the amino terminus of the sm aller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of Gen-Bank showed that the amino acid sequence of alpha-L-iduronidase ha s similarity to that of a bacterial beta-xylosidase. A full-length cDN A corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos -1 cells were transfected with pSVcIdu, their intracellular and secret ed level of alpha-L-iduronidase activity had increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs , which have no alpha-L-iduronidase activity, lacked the normal alpha- L-iduronidase mRNA of 2.2 kilobase and contained instead a trace amoun t of a 2.8-kilobase species. Isolation and characterization of an expr essible alpha-L-iduronidase cDNA represents the first step toward muta tion analysis and replacement therapy.