Lj. Stoltzfus et al., CLONING AND CHARACTERIZATION OF CDNA-ENCODING CANINE ALPHA-L-IDURONIDASE - MESSENGER-RNA DEFICIENCY IN MUCOPOLYSACCHARIDOSIS-I DOG, The Journal of biological chemistry, 267(10), 1992, pp. 6570-6575
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.