Specific Isoforms of the Resident Endoplasmic Reticulum Protein Glucosidase II Associate with the CD45 Protein-tyrosine Phosphatase via a Lectin-like Interaction

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic α-chain and an associated β-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the α-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIα is capable of associating with CD45 in a competitive situation where multiple GIIα isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIα acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation. We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic α-chain and an associated β-chain. To gain insight into the basis of the association between CD45 and GII, we examined the biochemical requirements for the interaction. We show that the α-subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GIIα is capable of associating with CD45 in a competitive situation where multiple GIIα isoforms are expressed. Further, transfection studies demonstrate that only isoforms containing the alternatively spliced sequence Box A1 are capable of binding CD45, although all isoforms are catalytically active. The interaction between CD45 and GII is dependent on the active site of GII, is mediated through the carbohydrate on CD45, and can be inhibited with mannose. Taken together, these results suggest that GIIα acts as a lectin and binds to CD45 in an exon-dependent manner. This lectin activity of GII may be a novel mechanism for the regulation of CD45 biology and play a role in immune function, possibly by regulating CD45 glycosylation. glucosidase II deoxynorjirmycin polyacrylamide gel electrophoresis endoplasmic reticulum endoglycosidase F CD45 is a highly abundant, transmembrane, protein-tyrosine phosphatase expressed on all cells of hematopoietic origin (1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (663) Google Scholar). The cytoplasmic phosphatase activity of CD45 has been shown to be essential for the early signal transduction events leading to both thymocyte maturation and T cell activation (1Trowbridge I.S. Thomas M.L. Annu. Rev. Immunol. 1994; 12: 85-116Crossref PubMed Scopus (663) Google Scholar). There is substantial evidence to suggest that CD45 regulates the tyrosine phosphorylation of Src family kinases (2Thomas M.L. Brown E.J. Immunol. Today. 1999; 20: 406-411Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 3Ashwell J.D. D'Oro U. Immunol. Today. 1999; 20: 412-416Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The external domain of CD45 is extremely heterogeneous with respect to size and carbohydrate content primarily because of three alternatively spliced exons that encode for potential O-linked glycosylations (4Thomas M.L. Lefrancois L. Immunol. Today. 1989; 9: 320-326Abstract Full Text PDF Scopus (145) Google Scholar). The usage of these exons appears to be developmentally regulated (4Thomas M.L. Lefrancois L. Immunol. Today. 1989; 9: 320-326Abstract Full Text PDF Scopus (145) Google Scholar). As well, the extracellular domain encodes attachment sites for numerous N-linked glycans, and these glycosylations appear to be important for cell surface expression and protein stability of CD45 (5Pulido R. Sanchez-Madrid F. Eur. J. Immunol. 1992; 22: 463-468Crossref PubMed Scopus (26) Google Scholar). Finally, although CD45 is a cell surface protein, no specific ligand for the extracellular domain has been definitively identified. Perhaps relevant to the present study, there have been studies suggesting that some lectins such as CD22 (6Stamenkovic I. Sgroi D. Aruffo A. Sy M.S. Anderson T. Cell. 1991; 66: 1133-1144Abstract Full Text PDF PubMed Scopus (314) Google Scholar,7Sgroi D. Koretzky G.A. Stamenkovic I. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4026-4030Crossref PubMed Scopus (81) Google Scholar), galectin-1 (8Pace K.E. Lee C. Stewart P.L. Baum L.G. J. Immunol. 1999; 163: 3801-3811Crossref PubMed Google Scholar, 9Perillo N.L. Pace K.E. Seilhamer J.J. Baum L.G. Nature. 1995; 378: 736-739Crossref PubMed Scopus (950) Google Scholar), and the mannan-binding protein (10Uemura K. Yokota Y. Kozutsumi Y. Kawasaki T. J. Biol. Chem. 1996; 271: 4581-4584Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) are able to bind to CD45 carbohydrate, although the biological significance of these interactions are largely not understood. Recently, our laboratory has demonstrated that the carbohydrate processing enzyme α-glucosidase II (GII)1 physically interacts with CD45 (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). GII is found within the ER and catalyzes the hydrolysis of the inner two α1,3-linked glucose residues present on allN-linked immature oligosaccharides (12Lucocq J.M. Brada D. Roth J. J. Cell Biol. 1986; 102: 2137-2146Crossref PubMed Scopus (74) Google Scholar, 13Hubbard S.C. Ivatt R.J. Annu. Rev. Biochem. 1981; 50: 555-583Crossref PubMed Scopus (892) Google Scholar). This processing of glucose in the ER has been shown to be intimately involved in protein folding by regulating the interaction between the nascent polypeptide and the lectin chaperones calnexin and calreticulin. More specifically, removal of the first α1,3-linked glucose by GII creates a substrate for calnexin/calreticulin binding (14Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (721) Google Scholar, 15Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 16Ora A. Helenius A. J. Biol. Chem. 1995; 270: 26060-26062Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 17Wada I. Kai M. Imai S. Sakane F. Kanoh H. EMBO J. 1997; 16: 5420-5432Crossref PubMed Scopus (79) Google Scholar), whereas removal of the second glucose causes dissociation of calnexin/calreticulin from the polypeptide (15Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (490) Google Scholar, 18Hebert D.N. Foellmer B. Helenius A. EMBO J. 1996; 15: 2961-2968Crossref PubMed Scopus (256) Google Scholar, 19Rodan A.R. Simons J.F. Trombetta E.S. Helenius E.S. EMBO J. 1996; 15: 6921-6930Crossref PubMed Scopus (139) Google Scholar, 20Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-199Abstract Full Text PDF PubMed Scopus (345) Google Scholar). The hydrolysis of the second α1,3-linked glucose is necessary for the progression of properly folded glycoproteins from the ER to the Golgi (21Sousa M. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (242) Google Scholar, 22Zapun A. Petrescu S.M. Rudd P.M. Dwek R.A. Thomas D.Y. Bergeron J.J.M. Cell. 1997; 88: 29-38Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The GII enzyme is composed of a 116-kDa α-subunit that contains a catalytic motif of the Family 31 glucosidases (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 23Flura T. Brada D. Ziak M. Roth J. Glycobiology. 1997; 7: 617-624Crossref PubMed Scopus (37) Google Scholar) and an 80-kDa β-chain of unknown function (24Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). We and others hypothesize that the β-chain is involved in enzyme localization (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Both subunits of GII have been shown to be alternatively spliced (25Arendt C.W. Dawicki W. Ostergaard H.L. Glycobiology. 1999; 9: 277-283Crossref PubMed Scopus (23) Google Scholar). There is one alternatively spliced sequence (Box B1) within GIIβ that gives rise to two potential isoforms, whereas within GIIα, there are two alternatively spliced sequences (Box A1 and A2) that have the potential to generate four distinct isoforms (25Arendt C.W. Dawicki W. Ostergaard H.L. Glycobiology. 1999; 9: 277-283Crossref PubMed Scopus (23) Google Scholar). These different splice forms may vary in their subcellular localization, enzymatic activity, or substrate specificity. The association between CD45 and GII may be surprising given the subcellular distribution and function of the two proteins; nevertheless, this interaction may prove to be instrumental in elucidating aspects of CD45 biology. Therefore, we wished to dissect the biochemical basis for this stable interaction. We found that only isoforms of GIIα containing Box A1 are capable of interacting with CD45. It also appears that the active site of GIIα is required for the interaction with the N-linked carbohydrate on CD45. As well, the addition of mannose significantly decreases the association between CD45 and GII. Together, these data suggest that the association between GII and CD45 is a lectin-based interaction. BW5147 (BW), and a CD45-negative variant (BW/T200−), mouse T-lymphoma cells were maintained as described previously (26Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1995; 270: 2313-2319Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The PHAr2.7 cell line, generously provided by Dr. Ian Trowbridge (Salk Institute, La Jolla, CA), is a BW-derived mutant deficient in GIIα subunit expression (23Flura T. Brada D. Ziak M. Roth J. Glycobiology. 1997; 7: 617-624Crossref PubMed Scopus (37) Google Scholar) that was maintained in an identical manner to the parental BW line. Monoclonal antibody I3/2.3, which was also provided by Dr. Ian Trowbridge, recognizes a pan-specific determinant within the CD45 extracellular region. I3/2.3 was purified and directly coupled to cyanogen-activated Sepharose 4B. Rabbit antiserum H2, specific for GIIβ, was described previously (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), whereas rabbit antiserum J37 was generated to the tandem intracellular phosphatase domains of CD45. The anti-GIIα antiserum was purchased from Stressgen (Vancouver, Canada). Complete sequences of clones 116FL.A (A1−/A2−), 116 FL.E (A1−/A2−), 116FL.B (A1+/A2−), and 6R5–14 (A1+/A2+) were obtained by the dideoxy chain termination method and compared with a previously published GII α-sequence (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The 116FL.A sequence contained no mutations, whereas mutations/truncations in the 116FL.B and 6R5–14 sequences were repaired by standard subcloning procedures. The 116FL.E and corrected 6R5–14 clones were then used to construct the A1-A2+ clone, and all four final clones were resequenced as above. Upon sequence verification, the cDNA fragments were cloned into the mammalian expression vector pcDNA3 (Invitrogen), and endotoxin-free preparations of the four constructs were prepared using the Qiagen EndoFree Plasmid Maxi kit. 20 μg of endotoxin-free DNA was electroporated into BW/PHAr cells using Bio-Rad Gene Pulser at 300 mV. Transfected cells were allowed to recover for 24–36 h, after which was to a final of were in of and for expression of GIIα by were at a of in and on for 20 were for with coupled or for with antiserum by a with protein were three with in and The was as described previously (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). CD45 were three in 20 to GII, by one in was then to the CD45 for h, by three with antibody at were on polyacrylamide and to as described previously (26Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1995; 270: 2313-2319Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). was with the by protein and by CD45 or GIIβ were prepared as described above. and the were in then allowed to the addition of of F The were then at for a was The were for at in of and of F The were by and or the were three with to the addition of and of the GII enzymatic activity was as described previously C.W. Ostergaard H.L. Glycobiology. PubMed Scopus Google Scholar). were with in for at was by the at as the obtained the was with was from all and were with to a of and were at were with the for at with of inhibited were and for GII activity as described above. Recently, was demonstrated that the BW5147 mutant cell BW/PHAr is deficient in expression of the GIIα subunit (23Flura T. Brada D. Ziak M. Roth J. Glycobiology. 1997; 7: 617-624Crossref PubMed Scopus (37) Google Scholar). we that BW/PHAr of CD45 and GIIβ We were able to the BW/PHAr cell to which GII subunit the interaction with CD45 that we have described previously (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). association of both GII subunits with CD45 is in the BW subunit associates with CD45 in the This two important of the expression of the GIIα subunit is required for the association of the GII with CD45. the directly with GIIβ associates with CD45 by of binding to This not the that GIIβ binding of GIIα to CD45 through a interaction or by a a in As there are four potential protein isoforms of GIIα that can be generated by (25Arendt C.W. Dawicki W. Ostergaard H.L. Glycobiology. 1999; 9: 277-283Crossref PubMed Scopus (23) Google Scholar). usage has not been demonstrated at the protein because of a of specific antibody the molecular weight of GIIα that with CD45 and the GIIα found in appears that a higher molecular weight of GIIα is found in association with CD45 This higher molecular weight is not in a of and is to a of we CD45 and GIIβ from BW a higher molecular weight of GIIα is in CD45 This is the that was CD45 and cell from BW cells The in of GIIα can from at two distinct the may be the of with a different size polypeptide and the may from such as glycosylation. To this CD45 and GIIβ were with F and by for The were with F enzyme the to the CD45 associated GIIα was with the to a form to the of the N-linked carbohydrate Therefore, all of the CD45 associated GIIα appears to be the GIIβ associated GIIα is with two distinct are two distinct polypeptide that or the spliced sequence (Box the data in appears that only a higher molecular weight form of GIIα associates with CD45. It is that isoforms of GIIα are capable of associating with CD45. To this we a CD45 were prepared for by with to GII. a was to the first CD45 that was then to the second CD45 This was for a of CD45 of the and after the was and examined for the of GIIα protein and GII enzymatic We found that only the higher molecular weight GIIα associated with CD45, and no molecular weight GIIα associated with CD45 after of the higher molecular weight form of GIIα the after the there was in the of the molecular weight of GIIα that there was of GIIα As well, there was of the GII enzymatic activity of activity the of Therefore, in a competitive situation where multiple GIIα isoforms are a higher molecular weight form of GIIα associates with CD45. The that only a higher molecular weight of GIIα is capable of association with CD45 the the molecular of this To this we transfection studies the BW/PHAr cell line. of the four isoforms of GIIα were generated in the BW/PHAr cells and used for CD45 from of the BW/PHAr and of the is that the association between CD45 and GII only in cells that GIIα isoforms containing the alternatively spliced sequence A1 whereas the second alternatively spliced (Box A2) not appear to the binding of GII to CD45 Therefore, the binding of GII to CD45 is by of Box all isoforms appear to have catalytic and of GII activity for the GII binding to CD45 not the data in GIIα protein is required for binding of GII to CD45. We to or not GIIα activity is necessary for CD45 binding by of active We a where CD45 from BW cells were of GII. To these we with a GII I. K. J. Biol. Chem. Full Text PDF PubMed Google Scholar), or a glucosidase R.J. M. 1989; PubMed Scopus Google Scholar). were to CD45 of GII, the of GII from that to bind CD45 was inhibited the no of GII binding to CD45 Further, of GII CD45 with not in a of GII binding not These data that to the of the an GII active site is can be these the there was a of there was of the GII activity in the all isoforms appear to have a enzymatic with not these data that the binding of GIIα to CD45 is inhibited at a of is the activity, which may a of GII for CD45 and a we demonstrated that to the association between CD45 and GII, carbohydrate on CD45 was required (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). in this we show that an active site on GII is also required to the association We then we the by To the carbohydrate to the we a that maintained the association in allowed F enzymatic Therefore, we a and CD45 with F. of CD45 with F in the of GII whereas the interaction These data that removal of the N-linked carbohydrate on both GII and CD45, GII is the interaction between CD45 and GII. This not between the of CD45 N-linked carbohydrate and the of GIIα N-linked our is to suggest that the N-linked carbohydrate on CD45 is important for the interaction (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This not the of GIIα N-linked carbohydrate to the the data in the association between CD45 and GII both the active site of GII and the CD45 N-linked These data suggest that the association between CD45 and GII is on a lectin interaction. from and J. Biol. Chem. Full Text PDF PubMed Google Scholar) demonstrated that to GII enzymatic activity, mannose residues are It is that GII also a mannose binding To the of or not GII a mannose binding function, we a where glucose or mannose were in the the addition of in the of GII to bind CD45, the addition of mannose significantly inhibited the binding of GII to CD45 As well, the addition of glucose or mannose no on the enzymatic activity of GII not Therefore, these data that the of mannose can the association of CD45 and GII and suggest that GII in mannose binding a we described the association between the protein-tyrosine phosphatase CD45 and the ER protein glucosidase II (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). This association appears to be extremely stable and has the to be in the (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). As well, to the association between CD45 and GII, there is a on carbohydrate on CD45 suggesting that GII is binding a interaction (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). present studies to the biochemical basis for the association between CD45 and GII in an to the potential of this association on CD45 biology. this we show that CD45 appears to with a higher molecular weight form of our transfection we found that only the Box and the isoforms are capable of binding CD45. Therefore, the alternatively spliced sequence A1 is necessary for the interaction of GIIα with CD45, whereas Box is for This is with our data obtained from the α-subunit purified on the basis of association with CD45, which demonstrated that the first alternatively spliced sequence was present (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). in suggest that the higher molecular weight GIIα is only a of GIIα found in the BW cell line. with chain of a of different cell that containing the first alternatively spliced were significantly the that this sequence (25Arendt C.W. Dawicki W. Ostergaard H.L. Glycobiology. 1999; 9: 277-283Crossref PubMed Scopus (23) Google Scholar). The that CD45 only associates with a of GIIα that there is a of to the interaction. This association is not because CD45 is an abundant, protein and because GII is an enzyme that is capable of this of the of this we have been able to an association of GII with N-linked containing such as (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), or A. and H. L. Taken together, these results suggest that CD45 associates with a Box of GII. of the for the first alternatively spliced sequence in GIIα, we that Box A1 to the association between CD45 and GII. on the Box A1 is not to the active site (11Arendt C.W. Ostergaard H.L. J. Biol. Chem. 1997; 272: 13117-13125Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 24Trombetta E.S. Simons J.F. Helenius A. J. Biol. Chem. 1996; 271: 27509-27516Abstract Full Text Full Text PDF PubMed Scopus (207) Google the of the protein may this sequence in to the active the enzymatic of GII in binding not glucose We this because all isoforms activity not GII is active not We that Box A1 GIIα with a novel binding activity or an binding Interestingly, we show that the addition of mannose can the of GII to with CD45. Therefore, Box GIIα not only a catalytic activity also a lectin This lectin activity is not of to GIIα to bind to mannose oligosaccharides because of from one of the GIIα with not in the binding GIIα to the not these we that both the active site and the lectin activity of Box A1 containing GIIα are required for the stable binding of GII to the N-linked carbohydrate on CD45. The of lectin activity in addition to the enzymatic activity within a carbohydrate processing is not Both and biochemical evidence in the for this of enzyme The between enzymatic activity and the lectin binding domain can be and in the lectin motif of not the activity of the enzyme B. R. D. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). in the of cell surface the activity of on the of lectin domain to bind D. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). Further, the of the enzyme can substrate binding and enzymatic activity lectin binding This is the for where substrate binding not binding J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, is that by both the GIIα active site and the there may be of one or both of the the stable association of GII with CD45. of the active site and the lectin domain may in a stable It is to that the association between CD45 and GII may have biological for CD45. GII may as a molecular that to CD45 within the ER to for the interaction with or regulated cell surface expression of CD45. The association with GII may also the enzymatic activity of CD45. in the phosphatase activity the of activity may the for As well, the stable interaction of GII with CD45 to on the carbohydrate of CD45, which CD45 where the association between CD45 and GII be of data suggest that the association not on the cell and because GII is an ER protein, we the interaction is at within the a function to ER To of the potential and binding of CD45 have been shown to with the carbohydrate on CD45, the biological significance of which is data suggest that GII is of a lectin that binds CD45. It is that CD45 not have one there are multiple that bind to CD45 carbohydrate and the function of CD45. We have to the biochemical basis for the and this has provided with insight into the biological to the interaction. These biological may to the role that the carbohydrate and extracellular CD45 in biology. As well, by GII interacts with we to gain into the role GII in the within the We Dr. Ian Trowbridge for the PHAr2.7 cell and We also Dr. for of this Dr. for and Dr. for the of the antiserum specific for the cytoplasmic domain of CD45.

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