Species-specific differences in the processing of acid α-glucosidase are due to the amino acid identity at position 201.

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R(200) and A(204) inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA(H201L) but not rhGAA(WT) to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA(H201L) but not rhGAA(WT) was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.

The alpha- and beta-subunits of the human UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase [corrected] are encoded by a single cDNA.

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an alpha2beta2gamma2-subunit structure was proposed. Although cDNA encoding the gamma-subunit has been described, cDNAs for the alpha- and beta-subunits have not. Using partial amino acid sequences from the bovine alpha- and beta-subunits, we have isolated a human cDNA that encodes both the alpha- and beta-subunits. Both subunits contain a single predicted membrane-spanning domain. The alpha- and beta-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the alpha/beta-subunits-precursor cDNA with or without the gamma-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the alpha-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-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 N-glycan 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.