Reduction of alpha-Gal expression by relocalizing alpha-galactosidase to the trans-Golgi network and cell surface.
Historically, the most effective means of modifying cell surface carbohydrates has required the intracellular overexpression of glycosyltransferases or glycosidases and is dependent on the enzymes occupying a cellular localization close to the carbohydrate structures they modify. We report on relocalizing the lysosomal resident glycosidase human alpha-galactosidase to other regions of the cell, Golgi and cell surface, where it is in closer proximity for cleaving the carbohydrate structure Galalpha(1,3)Gal. Relocalization of alpha-galactosidase was achieved by using the transmembrane and cytoplasmic domains from the human protein furin, which is known to localize in the trans-Golgi network (TGN) and cell surface. Two chimeric forms of alpha-galactosidase were generated, one directing it to the TGN of the cell and the other to the cell surface, as shown by confocal microscopy. The relocalized enzymes have the ability to cleave terminal alpha-galactose as detected by expression on the cell surface. Furthermore, when expressed as a transgene in mice, the TGN form of alpha-galactosidase was more effective at decreasing cell surface terminal alpha-galactose than was the native lysosomal form. When expressed in conjunction with the alpha1,2fucosyltransferase that also decreases Galalpha(1,3)Gal, the reduction was additive. The ability to relocalize enzymes that modify cell surface carbohydrate structures has far-reaching implications in biology and may be useful in such fields as xenotransplantation and treatment of glycosidase disorders.
Taylor
Simon G
SG
John Connell Laboratory of Glycobiology, Austin Research Institute, Austin, USA.
Osman
Narin
N
McKenzie
Ian F C
IF
Sandrin
Mauro S
MS
D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000201:enzymology*; D045683:Furin / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000201:enzymology / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D000519:alpha-Galactosidase; D045683:Furin; D005690:Galactose