Mucopolysaccharidosis type IIIB (
MPS-IIIB, Sanfilippo type B Syndrome) is a heterosomal, recessive lysosomal storage disorder resulting from a deficiency of [
alpha]-N-acetylglucosaminidase (
NAGLU). To characterize this
enzyme further and evaluate its potential for
enzyme replacement studies we expressed the
NAGLU-encoding
cDNA in Chinese hamster ovary cells (CHO-K1 cells) and purified the recombinant
enzyme from the medium of stably transfected cells by a two-step affinity chromatography. Two
isoforms of recombinant
NAGLU with apparent molecular weights of 89 and 79 kDa were purified and shown to differ in their glycosylation pattern. The catalytic parameters of both forms of the recombinant
enzyme were indistinguishable from each other and similar to those of
NAGLU purified from various tissues. However, compared to other recombinant lysosomal
enzymes expressed from CHO-K1 cells, the
mannose-6-phosphate receptor mediated uptake of the secreted form of recombinant
NAGLU into cultured skin fibroblasts was considerably reduced. A small amount of phosphorylated
NAGLU present in purified
enzyme preparations was shown to be endocytosed by
MPS-IIIB fibroblasts via the
mannose-6-phosphate receptor-mediated pathway and transported to the lysosomes, where they corrected the storage phenotype. Direct metabolic labeling experiments with Na(2) (32)PO(4) confirmed that the specific phosphorylation of recombinant
NAGLU secreted from transfected CHO cells is significantly lower when compared with a control lysosomal
enzyme. These results suggest that the use of secreted
NAGLU in future
enzyme and gene replacement
therapy protocols will be severely limited due to its small degree of mannose-6-phosphorylation.