Lysosomal Acid α-Glucosidase Consists of Four Different Peptides Processed from a Single Chain Precursor

Abstract

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...

Antibody-mediated enzyme replacement therapy targeting both lysosomal and cytoplasmic glycogen in Pompe disease

“…1b). Western blotting on L6 cell lysates using an anti-GAA antibody revealed that in addition to the typical 95-and 76 kDa processed (lysosomal) forms that are also seen in the rhGAA-treated cells [29], there existed a 150 kDa fulllength FabGAA, presumably the cytoplasmic form, in the FabGAA-treated cells (Fig. 1c).…”

Silent exonic mutation in the acid-α-glycosidase gene that causes glycogen storage disease type II by affecting mRNA splicing

“…Moreover, a small polypeptide (amino acids 57-78) has been shown to undergo cleavage during GAA maturation. 4 However, several other pathogenic mutations in the GAA gene have been reported to be located in the regions that are proteolytically removed during the maturation of the enzyme. 7 Therefore, further functional studies may be required to clarify the association between this mutation and GSDII.…”

“…2 The GAA cDNA contains a 2856-bp-long coding sequence and encodes a 952-amino-acid polypeptide with a calculated molecular mass of 105 kDa. 3,4 The enzyme is synthesized as a catalytically inactive precursor and undergoes posttranslational modification. 5,6 The precursor protein then undergoes a stepwise proteolysis at both termini, yielding the mature GAA consisting of four associated polypeptides.…”

“…5,6 The precursor protein then undergoes a stepwise proteolysis at both termini, yielding the mature GAA consisting of four associated polypeptides. 4 We conducted a molecular genetic study of a Japanese patient with childhood-onset GSDII. The patient was found to be a compound heterozygote for a recurrent missense mutation and for a newly discovered splice-site mutation of the GAA gene.…”

Biochemical and structural study on a S529V mutant acid α-glucosidase responsive to pharmacological chaperones

“…Finally, it is transported to lysosomes, where it is modified into a 76 kDa mature enzyme and a minor 70 kDa component via a 95 kDa intermediate. [1][2][3][4] A genetic defect in the enzyme causes the accumulation of glycogen in lysosomes and an abnormality of autophagy, 5 thereby leading to Pompe disease (glycogen storage disease type II; OMIM 232300). The disease exhibits a wide range of clinical phenotypes, from the earlyonset severe 'infantile form' , which is characterized by cardiomegaly, hepatomegaly, hypotonia and muscular weakness, to the late-onset slowly progressive 'juvenile/adult form' , which shows a predominance of muscular disorders.…”