beta-glucocerebrosidase, the enzyme defective in Gaucher disease, is targeted to the lysosome independently of the mannose-6-phosphate receptor. Affinity-chromatography experiments revealed that the lysosomal integral membrane protein LIMP-2 is a specific binding partner of beta-glucocerebrosidase. This interaction involves a coiled-coil domain within the lumenal domain. beta-glucocerebrosidase activity and protein levels were severely decreased in LIMP-2-deficient mouse tissues. Analysis of fibroblasts and macrophages isolated from these mice indicated that the majority of beta-glucocerebrosidase was secreted. Missorting of beta-glucocerebrosidase was also evident in vivo, as protein and activity levels were significantly higher in sera from LIMP-2-deficient mice compared to wild-type. Reconstitution of LIMP-2 in LIMP-2-deficient fibroblasts led to a rescue of beta-glucocerebrosidase levels and distribution. LIMP-2 expression also led to lysosomal transport of a beta-glucocerebrosidase endoplasmic reticulum retention mutant. These data support a role for LIMP-2 as the mannose-6-phosphate-independent trafficking receptor for beta-glucocerebrosidase.

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Pompe's disease is caused by a deficiency of the lysosomal enzyme acid ␣-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and Nglycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78 -113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204 -782) contains the 3.9-and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.Lysosomal acid ␣-glucosidase (GAA 1 ; EC 3.2.1.3) is an exo-1,4-and -1,6-␣-glucosidase that hydrolyzes glycogen to glucose. The cDNA for GAA encodes a protein of 952 amino acids with a predicted molecular mass of 105 kDa (1). The newly synthesized precursor has an amino-terminal signal peptide for cotranslational transport into the lumen of the endoplasmic reticulum, where it is N-glycosylated at seven glycosylation sites, resulting in a glycosylated precursor with an apparent molecular mass of 110 kDa.The intracellular processing of GAA has been investigated previously (2, 3). It was proposed that, after transport through the Golgi complex and targeting to the endosome/lysosome, the 110-kDa precursor is proteolytically processed at the amino terminus, resulting in a 95-kDa intermediate with a sequence beginning at amino acid 122. Prior to this study, the 95-kDa intermediate was proposed to be proteolytically processed to a 76-kDa form, which was believed to occur between amino acids 816 and 881 (3). The 76-kDa form is then proteolytically processed at the amino terminus at amino acid 204 to give the 70-kDa mature form (3). The nomenclature used for the processed forms of GAA is based on apparent molecular mass as determined by SDS-PAGE.The identities of the proteases involved in the maturation of GAA have never been established. GAA has been purified from many different tissues such as bovine testis (4), rat liver (5), pig liver (6), human liver (7), rabbit mus...

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Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO-cell derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO-cell derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.

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