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.
Moreland
Rodney J
RJ
Genzyme Corporation, Oklahoma City, Oklahoma 73104, USA.
Jin
Xiaoying
X
Zhang
X Kate
XK
Decker
Roger W
RW
Albee
Karen L
KL
Lee
Karen L
KL
Cauthron
Robert D
RD
Brewer
Kevin
K
Edmunds
Tim
T
Canfield
William M
WM
D000595:Amino Acid Sequence; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D005087:Glucan 1,4-alpha-Glucosidase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D006009:Glycogen Storage Disease Type II / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D009097:Multienzyme Complexes; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D011498:Protein Precursors / Q000378:metabolism*; D021122:Protein Subunits; D017527:Receptor, IGF Type 2 / Q000502:physiology; D016415:Sequence Alignment; D000520:alpha-Glucosidases
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011498:Protein Precursors; D021122:Protein Subunits; D017527:Receptor, IGF Type 2; D000520:alpha-Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase; D010447:Peptide Hydrolases