Identification of the CD45-associated 116-kDa and 80-kDa Proteins as the α- and β-Subunits of α-Glucosidase II

Abstract

CD45 is an abundant, highly glycosylated transmembrane protein-tyrosine phosphatase expressed on hematopoietic cells. Herein we demonstrate that two proteins of 116 kDa and 80 kDa copurify with CD45 from mouse T cells. Microsequence analysis of the 116-kDa protein revealed high similarity to an incomplete human open reading frame that has been suggested to correspond to the catalytic ␣-subunit of glucosidase II. We determined the nucleotide sequence of the mouse cDNA and observed that it encodes a protein product nearly identical to its human homologue and shares an active site consensus sequence with Family 31 glucosidases. Amino acid sequencing of the 80-kDa protein, followed by molecular cloning, revealed high homology to human and bovine cDNAs postulated to encode the ␤-subunit of glucosidase II. Antisera developed to the mouse ␤-subunit allowed us to demonstrate that the interaction between CD45 and glucosidase II can be reconstituted in vitro in an endoglycosidase H-sensitive manner. The strong interaction between glucosidase II and CD45 may provide a paradigm for investigating novel aspects of the biology of these proteins.CD45 is a high molecular mass (ϳ180-ϳ220 kDa) transmembrane protein-tyrosine phosphatase (PTP) 1 expressed in abundance on all cells of hematopoietic lineage (1). Although CD45 is encoded by a single gene, alternative splicing of the mRNA allows for the generation of at least eight distinct isoforms of the molecule based on variable usage of exons 4 -6, which encode a region of the amino-terminal ectodomain. Of particular interest, and experimental utility, is the observation that these isoforms are expressed in a cell type-specific and differentiation stage-specific manner. The variable exon repertoire expressed by different isoforms is known to greatly influence the charge properties of CD45 since the region encoded by these exons contains multiple sites for O-linked glycosylation (2). CD45 also possesses 11-18 putative N-linked glycosylation sites, which are subject to cell-specific controls, conferring further microheterogeneity upon CD45 (3). It has been estimated that approximately one third of the total molecular weight of mature CD45 is contributed by carbohydrates, which vary qualitatively and quantitatively depending on the particular cell and isoform(s) expressed (3).The cytoplasmic domain of CD45 is identical among all isoforms and encodes two tandem PTP domains, at least one of which possesses intrinsic activity (1). A series of studies in CD45-deficient cell lines (1) and, more recently, in CD45 genedisrupted mice (4, 5) have revealed that CD45 functions as a key regulator of maturation and activation pathways in lymphocytes. One mechanism through which CD45 appears to function is by regulating the activity of various members of the Src family of protein-tyrosine kinases (1). Despite the advances that have been made, questions remain regarding many aspects of the biology of CD45. For example, it is not known whether certain, as yet unidentified, factors are responsible for...

Structure, Properties, and Tissue Localization of Apoplastic α-Glucosidase in Crucifers1

“…Sequences from ESTs derived from Aglu-2 strongly suggest that it is also a member of clade 1. Aglu-3 fell into clade 2 along with the ER glucosidase II from mouse (Arendt and Ostergaard, 1997) and several sequenced eukaryotic genes, including a cDNA from potato (Taylor et al, 1998). Partial amino acid sequence from the ER glucosidase II purified from rat liver closely matches the mouse glucosidase II sequence (Trombetta et al, 1996).…”

“…The ER glucosidase II is a distant relative of this family (Trombetta et al, 1996;Arendt and Ostergaard, 1997). ESTs from several different Arabidopsis genes were identified, and one, 38A2T7 (accession no.…”

Developmentally Regulated Changes in Glucosidase II Association with, and Carbohydrate Content of, the Protein Tyrosine Phosphatase CD45

“…The hybridoma secreting the class I MHC-specific mAb M1/42.3.9.8 was purchased from American Type Culture Collection (Manassas, VA). Rabbit antisera H2 and J37, specific to GII␤ and the intracellular region of CD45, respectively, were previously described (20,24). Rabbit antiserum L177 was generated to a peptide fragment corresponding to the alternatively spliced Box A1 region of GII␣ coupled to keyhole limpet hemocyanin (25).…”

Cloning and Characterization of the Glucosidase II Alpha Subunit Gene of Trichoderma reesei : a Frameshift Mutation Results in the Aberrant Glycosylation Profile of the Hypercellulolytic Strain Rut-C30

“…Since then, glucosidase II has been partially or completely purified from a wide variety of both lower and higher eukaryotic organisms (2,6,7,11,20,38,78,80,88). Following some initial controversy, it is now generally accepted that the protein is a rather asymmetric nonglobular heterodimer consisting of a catalytic alpha subunit with a molecular mass of about 110 kDa (GII␣) and a beta subunit with a molecular mass of 60 kDa (GII␤) (6,15,65,85,86,88).…”

“…Disruption of the latter ORF yielded a yeast strain devoid of glucosidase II activity (88). Shortly after this, GII␣-encoding sequences were cloned from mouse T lymphocytes (6), pig liver (20), the slime mold Dictyostelium discoideum (22), and Schizosaccharomyces pombe (15). All derived protein sequences carry a conserved WX DLNE motif, which is the typical consensus sequence for the active site of glycosidase family 31 (29).…”