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J. Biol. Chem., Vol. 275, Issue 41, 32071-32076, October 13, 2000

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

Troy A. Baldwin, Markéta Gogela-Spehar, and Hanne L. Ostergaard

From the Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, T6G 2S2 Alberta, Canada

Received for publication, April 11, 2000, and in revised form, July 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have previously demonstrated that CD45 physically associates with the endoplasmic reticulum processing enzyme glucosidase II (GII). GII consists of the catalytic -chain and an associated -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 -subunit is essential for the interaction. Interestingly, only a higher molecular weight form of GII is capable of associating with CD45 in a competitive situation where multiple GII 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 GII 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CD45 is a highly abundant, transmembrane, protein-tyrosine phosphatase expressed on all cells of hematopoietic origin (1). 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 (1). There is substantial evidence to suggest that CD45 regulates the tyrosine phosphorylation of Src family kinases (2, 3). 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 (4). The usage of these exons appears to be developmentally regulated (4). 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 (5). 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 (6, 7), galectin-1 (8, 9), and the mannan-binding protein (10) 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 -glucosidase II (GII)¹ physically interacts with CD45 (11). GII is found within the ER and catalyzes the hydrolysis of the inner two 1,3-linked glucose residues present on all N-linked immature oligosaccharides (12, 13). 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 1,3-linked glucose by GII creates a substrate for calnexin/calreticulin binding (14-17), whereas removal of the second glucose causes dissociation of calnexin/calreticulin from the polypeptide (15, 18-20). The hydrolysis of the second 1,3-linked glucose is necessary for the progression of properly folded glycoproteins from the ER to the Golgi (21, 22).

The GII enzyme is composed of a 116-kDa -subunit that contains a catalytic motif of the Family 31 glucosidases (11, 23) and an 80-kDa -chain of unknown function (24). We and others hypothesize that the -chain is involved in enzyme localization (11, 24). Both subunits of GII have been shown to be alternatively spliced (25). There is one alternatively spliced sequence (Box B1) within GII that gives rise to two potential isoforms, whereas within GII, there are two alternatively spliced sequences (Box A1 and A2) that have the potential to generate four distinct isoforms (25). 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 GII containing Box A1 are capable of interacting with CD45. It also appears that the active site of GII 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Lines and Antibody Reagents-- BW5147 (BW), and a CD45-negative variant (BW/T200), mouse T-lymphoma cells were maintained as described previously (26). The PHA^r2.7 cell line, generously provided by Dr. Ian Trowbridge (Salk Institute, La Jolla, CA), is a BW-derived mutant deficient in GII subunit expression (23) 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 GII, was described previously (11), whereas rabbit antiserum J37 was generated to the tandem intracellular phosphatase domains of CD45. The anti-GII antiserum was purchased from Stressgen (Vancouver, Canada).

Establishment of BW/PHA^r Cell Lines Expressing Individual GII Isoforms-- 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 -sequence (11). 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/PHA^r cells using Bio-Rad Gene Pulser at 300 mV. Transfected cells were allowed to recover for 24-36 h, after which G-418 (Life Technologies) was added to a final concentration of 2 mg/ml. Selected transfectants were grown up in presence of G-418 and screened for expression of GII by Western blotting.

Cell Lysis, Immunoprecipitation, and Reconstitution Assays-- Cells were lysed at a density of 5 × 10⁷/ml in 0.5% Nonidet P-40 (Pierce), 150 mM NaCl, 10 mM Tris, pH 7.6, and incubated on ice for 20 min. Post nuclear supernatants were incubated for 1-2 h with I3/2 coupled beads or for 20-30 min with polyclonal antiserum followed by a 1-2 h incubation with protein A-Sepharose beads (Roche Molecular Biochemicals). Immunoprecipitates were washed three times with ice-cold lysis buffer, resuspended in reducing sample buffer, and boiled. The reconstitution assay was performed as described previously (11). Briefly, CD45 immunoprecipitates were washed three times in 0.5% deoxycholate, 20 mM Tris, pH 7.6, to remove bound GII, followed by one wash in lysis buffer. A BW/T200 lysate was then added to the CD45 immunoprecipitate for 1-2 h, followed by three washes with lysis buffer. All antibody incubations took place at 4 °C.

Polyacrylamide Gel Electrophoresis and Immunoblotting-- Proteins were resolved on 7.5% polyacrylamide gels and transferred to polyvinylidene difluoride-Immobilon (Millipore) as described previously (26). Western blot analysis was carried out with the indicated antiserum, followed by protein A-horseradish peroxidase (Pierce), and visualized by enhanced chemiluminescence (PerkinElmer Life Sciences).

Endoglycosidase Treatment-- CD45 or GII immunoprecipitates were prepared as described above. For treatment under reducing and denaturing conditions, the immunoprecipitates were boiled in 1% Nonidet P-40, 0.1% SDS, 1% -mercaptoethanol, 10 mM phosphate, 150 mM NaCl, pH 7.2, then allowed to cool before the addition of 0.3 unit of Endo F (Sigma). The beads were then incubated at 33 °C for 16-18 h. For treatment under native conditions a 50 mM imidazole pH 6.8 buffer was used. The beads were incubated for 16-18 h at 33 °C in 50 µl of imidazole buffer and 0.3 unit of Endo F (Sigma). The reactions were quenched by adding reducing sample buffer and boiling, or the beads were washed three times with lysis buffer prior to the addition of reducing sample buffer and boiling.

GII Enzymatic Assay-- Determination of the GII enzymatic activity was performed as described previously (27). Briefly, samples were incubated with 5 mM p-nitrophenyl -D-gluocopyranoside (Sigma) in phosphate-buffered saline, pH 7.2, for 16-18 h at room temperature. Color change was quantified by measuring the absorbance at 405 nm. Background absorbance, defined as the average value obtained when the colorimetric reagent was incubated with lysis buffer alone, was subtracted from all values obtained.

Inhibitor Treatment of Lysates-- Deoxynorjirmycin (dNM) and australine (Oxford Glycosystems, Wakefield, MA) were reconstituted with water to a concentration of 100 mM, and aliquots were stored at 20 °C. Lysates were treated with the inhibitors for 1 h at room temperature with rotation. Samples of inhibited lysates were kept and analyzed for GII activity as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The CD45-GII Interaction Requires the GII Subunit-- Recently, it was demonstrated that the BW5147 mutant cell line BW/PHA^r is deficient in expression of the GII subunit (23). However, we find that BW/PHA^r expresses normal levels of CD45 and GII (Fig. 1). We were therefore able to utilize the BW/PHA^r cell line to determine which GII subunit mediates the interaction with CD45 that we have described previously (11). An association of both GII subunits with CD45 is observed in the BW cells, but neither subunit associates with CD45 in the BW/PHA^r cells (Fig. 1). This result demonstrates two important features of the CD45-GII association. First, expression of the GII subunit is required for the association of the GII complex with CD45. Second, the -subunit alone cannot interact directly with CD45; rather, GII associates with CD45 by virtue of its binding to GII. This observation does not preclude the possibility that GII might enhance binding of GII to CD45 perhaps through a protein-protein interaction or by a causing a conformational change in GII.

View larger version (54K): [in this window] [in a new window]   Fig. 1.   GII protein expression is required for the association with CD45. I3/2 beads were incubated with 2.5 × 107 BW or BW/PHAr post-nuclear extracts, and the immunoprecipitates were resolved by SDS-PAGE. Western blotting (WB) for the presence of GII (top panel), GII (middle panel), or CD45 (bottom panel) was performed. Whole cell lysate (WCL) from the indicated cell type was probed for protein expression.

A Minor, Higher Molecular Weight Isoform of GII Associates with CD45-- As stated previously, there are four potential protein isoforms of GII that can be generated by alternative splicing (25). Variable isoform usage has not yet been conclusively demonstrated at the protein level, mainly because of a lack of specific antibody reagents. In comparing the molecular weight of GII that co-immunoprecipitates with CD45 and the total GII pool found in lysates, it appears that a higher molecular weight isoform of GII is found in association with CD45 (Fig. 1). This higher molecular weight isoform is not readily detectable in a Western blot of total lysate and is therefore thought to constitute a minor portion of total GII. When we compare CD45 and GII immunoprecipitates from BW cells, a higher molecular weight isoform of GII is observed in CD45 immunoprecipitates (Fig. 2). This is the same pattern that was observed when comparing CD45 immunoprecipitates and total cell lysate from BW cells (Fig. 1). The difference in relative mobility of GII subsets can arise from at least two distinct mechanisms. First, the difference may be the result of alternative splicing with a different size polypeptide backbone, and second, the difference may arise from post-translational modifications such as differential glycosylation. To address this issue, CD45 and GII immunoprecipitates were digested with Endo F under reducing and denaturing conditions, followed by Western blot analysis for GII. The samples were spiked with additional Endo F enzyme periodically throughout the incubation to achieve complete digestion. When the CD45 associated GII was treated with Endo F, the entire band shifted to a faster migrating form owing to the release of the N-linked carbohydrate (Fig. 2). Therefore, all of the CD45 associated GII appears to be glycosylated. When the GII associated GII is digested with Endo F, two distinct bands are revealed (Fig. 2) likely representing two distinct polypeptide backbones that contain or lack the larger differentially spliced sequence (Box A1).

View larger version (49K): [in this window] [in a new window]   Fig. 2.   A higher molecular weight isoform of GII associates with CD45. CD45 or GII was immunoprecipitated (i.p.) from 2.5 × 107 BW cell lysates followed by mock treatment, or treatment with 0.3 unit of Endo F under reducing and denaturing conditions. Proteins were resolved by SDS-PAGE and immunoblotted for either GII (upper panel) or CD45 (lower panel). WB, Western blotting.

Only a Higher Molecular Weight Isoform of GII Is Capable of Associating with CD45-- From the data presented in Fig. 2, it appears that only a higher molecular weight form of GII associates with CD45. It is possible, however, that other isoforms of GII are capable of associating with CD45. To address this issue, we employed a sequential reconstitution strategy. First, CD45 immunoprecipitates were prepared for reconstitution assays by washing with 0.5% deoxycholate to remove endogenously bound GII. Next, a BW/T200 lysate was added to the first CD45 immunoprecipitate. After incubation, that same lysate was then added to the second CD45 immunoprecipitation. This was repeated for a total of six CD45 immunoprecipitates. A sample of the BW/T200 lysate before and after the sequential reconstitution assays was taken and examined for the presence of GII protein and GII enzymatic activity. We found that only the higher molecular weight GII associated with CD45, and no lower molecular weight GII associated with CD45 even after depletion of the higher molecular weight form of GII (Fig. 3). In examining the lysate after the sequential reconstitutions, there was little change in the amount of the lower molecular weight isoform of GII (Fig. 3), demonstrating that there was still plenty of GII available. As well, there was still 75% of the initial GII enzymatic activity remaining, so loss of activity cannot explain the lack of binding. Therefore, in a competitive situation where multiple GII isoforms are expressed, a higher molecular weight form of GII preferentially associates with CD45.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Only a higher molecular weight isoform of GII is capable of associating with CD45. CD45 immunoprecipitates from 2.5 × 107 BW post nuclear extracts were washed with 0.5% deoxycholate, 20 mM Tris, pH 7.6, to remove bound GII. A lysate corresponding to 1.5 × 107 cell equivalents of BW/T200 was added sequentially to the washed CD45 immunoprecipitates. Beads were washed three times in lysis buffer and subjected to SDS-PAGE analysis. Western blot (WB) analysis was performed for GII (top panel), GII (middle panel), and CD45 (bottom panel). Whole cell lysate (WCL) before (pre-rec.) and after (post-rec.) the sequential reconstitutions was also analyzed for the presence of the indicated proteins and GII enzymatic activity.

Only Isoforms of GII Containing Alternatively Spliced Sequence A1 Are Capable of Associating with CD45-- The observation that only a higher molecular weight isoform of GII is capable of association with CD45 raises the obvious question regarding the molecular identity of this isoform. To answer this question, we performed transfection studies utilizing the BW/PHA^r cell line. Stable transfectants expressing each of the four isoforms of GII were generated in the BW/PHA^r cells and used for immunoprecipitation experiments. In examining CD45 immunoprecipitates from lysates of the BW, BW/PHA^r parentals and each of the transfectants, it is clear that the association between CD45 and GII only occurs in cells that express GII isoforms containing the alternatively spliced sequence A1 (Fig. 4), whereas the second alternatively spliced region (Box A2) does not appear to influence the binding of GII to CD45 (Fig. 4). Therefore, the binding of GII to CD45 is dictated by presence of Box A1. Of note, all isoforms appear to have similar catalytic activities, and therefore, alteration of GII activity cannot account for the isoform-specific GII binding to CD45 (data not shown).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Only the GII isoforms containing Box A1 are capable of associating with CD45. CD45 or GII immunoprecipitates were prepared from the post nuclear extracts of 2.5 × 107 BW; BW/PHAr; or the BW/PHAr +/+ (BoxA1+/Box A2+), +/ (Box A1+/Box A1), /+ (Box A1/Box A2+), / (Box A1/Box A2) transfectants. Either the immunoprecipitates (ip.) were resolved by SDS-PAGE or a GII enzymatic activity assay was carried out. Western blot (WB) analysis for GII, GII, and CD45 was performed.

The GII Catalytic Site Is Essential for the Binding of GII to CD45-- From the data presented in Fig. 1, GII protein is required for binding of GII to CD45. We next wanted to determine whether or not GII activity is necessary for CD45 binding by making use of competitive, active site-directed inhibitors. We performed a reconstitution assay where CD45 immunoprecipitates from BW cells were stripped of endogenously bound GII. To these immunoprecipitates, we added BW/T200 lysates pretreated with either dNM, a GII inhibitor (28), or australine, a glucosidase I-specific inhibitor (29). When dNM-treated lysates were added to CD45 immunoprecipitates devoid of GII, the ability of GII from that lysate to bind CD45 was inhibited (Fig. 5A). On the other hand, australine-treated lysates showed no impairment of GII binding to CD45 (Fig. 5A). Further, treatment of GII bound CD45 immunoprecipitates with dNM, but not australine, resulted in a loss of GII binding (data not shown). These data indicate that to initiate the formation of the CD45-GII complex, an unoccupied GII active site is required. Another interesting point can be made regarding these data. In the 0.01 mM dNM-treated lysate, there was almost a complete inhibition of reconstitution; yet there was still 81% of the GII activity remaining in the lysate (Fig. 5A). Because all isoforms appear to have a similar enzymatic inhibition curve with dNM (data not shown), these data indicate that the binding of GII to CD45 is inhibited at a lower concentration of inhibitor than is the activity, which may reflect a differential affinity of GII for CD45 and substrate.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   An unoccupied GII active site and CD45 N-linked carbohydrate is required for the CD45-GII association. I3/2 coupled beads were incubated with extracts from 2.5 × 107 BW cells and washed with 0.5% deoxycholate, 20 mM Tris, pH 7.6, to remove bound GII. BW/T200 lysates were incubated with the indicated concentration of dNM or australine (aust.) for 1 h at room temperature. The inhibited lysates were mixed with the deoxycholate-washed CD45 immunoprecipitates for 2 h at 4 °C. The ability of GII from the inhibited lysate to reassociate with CD45 was assessed by Western blot (WB) analysis for GII (top panel), GII (middle panel), and CD45 (bottom panel). A sample of the inhibitor treated lysate was analyzed for GII activity by the colorimetric assay described under "Experimental Procedures" and reported as a percentage of the untreated lysate. B, CD45 immunoprecipitates from 2.5 × 107 BW post nuclear lysates were either untreated (mock), or treated with Endo F under native conditions for 16 h at 33 °C. All immunoprecipitates (ip.) were washed with lysis buffer prior to SDS-PAGE analysis. Immunoblotting was performed for GII (top panel), GII (middle panel), and CD45 (bottom panel). WB, Western blotting.

Removal of CD45 N-linked Carbohydrate Can Disrupt the Preformed CD45-GII Association-- In a previous report, we demonstrated that to reconstitute the association between CD45 and GII, Endo H-sensitive carbohydrate on CD45 was required (11). In addition, in this report, we show that an unoccupied active site on GII is also required to reconstitute the association (Fig. 5A). We then asked whether we could disrupt the preformed complex by removing the N-linked carbohydrate. To determine the contribution of N-linked carbohydrate to the association, we needed a buffering system that maintained the association in native form, yet allowed Endo F enzymatic activity. Therefore, we utilized a 50 mM imidazole pH 6.8 buffer and treated CD45 immunoprecipitates with Endo F. Treatment of CD45 immunoprecipitates with Endo F resulted in the loss of GII association, whereas mock treatment preserved the interaction (Fig. 5B). These data indicate that removal of the N-linked carbohydrate on both GII and CD45, while GII is bound, will disrupt the interaction between CD45 and GII. This experiment does not discriminate between the contribution of CD45 N-linked carbohydrate and the contribution of GII N-linked carbohydrate. Given our previous reconstitution experiments, however, it is reasonable to suggest that the N-linked carbohydrate on CD45 is important for the interaction (11). This does not preclude the possibility of GII N-linked carbohydrate contributing to the association.

The Addition of Mannose Prevents Reconstitution of the Interaction between CD45 and GII-- From the data presented in Fig. 5, the association between CD45 and GII requires both the active site of GII and the CD45 N-linked carbohydrate. These data suggest that the association between CD45 and GII is based on a lectin interaction. A report from Grinna and Robbins (30) demonstrated that to obtain optimal GII enzymatic activity, branched mannose residues are required. It is therefore possible that GII also possess a mannose binding activity. To address the issue of whether or not GII does indeed possess a mannose binding function, we performed a reconstitution assay where either glucose or mannose monosaccharides were included in the reconstitution assay. Although the addition of 10 mM glucose caused little change in the ability of GII to bind CD45, the addition of 10 mM mannose significantly inhibited the binding of GII to CD45 (Fig. 6). As well, the addition of either glucose or mannose had no effect on the enzymatic activity of GII (data not shown). Therefore, these data indicate that the presence of mannose can inhibit the association of CD45 and GII and suggest that GII does in fact possess mannose binding activity.

View larger version (38K): [in this window] [in a new window]   Fig. 6.   The addition of mannose inhibits the reassociation of GII with CD45. I3/2 coupled beads were incubated with extracts from 2.5 × 107 BW cells and washed with 0.5% deoxycholate, 20 mM Tris, pH 7.6, to remove bound GII. BW/T200 lysates containing the indicated concentration of monosaccharide was incubated with the washed CD45 immunoprecipitates for 2 h at 4 °C. The beads were then washed three times with lysis buffer prior to analysis by SDS-PAGE. Western blotting (WB) was performed for GII (top panel), GII (middle panel), and CD45 (bottom panel). No inhibition of GII enzymatic activity by the addition of the monosaccharides was observed. glc., glucose; man., mannose.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In a previous report, we described the physical association between the protein-tyrosine phosphatase CD45 and the resident ER protein glucosidase II (11). This association appears to be extremely stable and has the ability to be reconstituted in vitro under the appropriate conditions (11). As well, to reconstitute the association between CD45 and GII, there is a dependence on Endo H-sensitive carbohydrate on CD45 suggesting that GII is binding via a lectin-like interaction (11). Our present studies sought to further elucidate the biochemical basis for the association between CD45 and GII in an attempt to understand the potential implications of this association on CD45 biology.

In this report, we show that CD45 appears to strongly interact with a minor, higher molecular weight form of GII. Through our transfection studies, we found that only the Box A1+/A2+ and the A1+/A2 isoforms are capable of binding CD45. Therefore, the alternatively spliced sequence A1 is necessary for the interaction of GII with CD45, whereas Box A2 is dispensable for binding. This finding is consistent with our amino acid sequencing data obtained from the -subunit purified on the basis of its association with CD45, which demonstrated that the first alternatively spliced sequence was present (11). Data presented in Fig. 3 suggest that the higher molecular weight CD45-interacting GII is only a relatively minor subset of total GII found in the BW cell line. Consistent with this, reverse transcription-polymerase chain reaction analysis of a number of different cell lines indicated that transcripts containing the first alternatively spliced exon were significantly less abundant than the transcripts that lacked this sequence (25). The fact that CD45 only associates with a minor population of GII indicates that there is a high degree of specificity to the interaction. This association is not taking place simply because CD45 is an abundant, carbohydrate-containing protein and because GII is an enzyme that is capable of modifying this carbohydrate. In further support of the specificity of this interaction, we have never been able to detect an association of GII with other abundant N-linked glycan containing proteins such as Class I major histocompatibility complex (11), LFA-1, or CD44.² Taken together, these results suggest that CD45 specifically associates with a minor Box A1-containing subset of GII.

Because of the requirement for the first alternatively spliced sequence in GII, we posit that Box A1 functions to stabilize the association between CD45 and GII. Based on the primary sequence, Box A1 is not adjacent to the active site (11, 24); however, the tertiary structure of the protein may place this sequence in close proximity to the active site, thereby modifying the enzymatic properties of GII resulting in binding but not glucose cleavage. We find this possibility unlikely because all isoforms contain equal activity (data not shown). Furthermore, CD45-associated GII is fully active (data not shown). We find it more likely that Box A1 could provide GII with a novel binding activity or stabilize an existing binding activity. Interestingly, we show that the addition of mannose can inhibit the ability of GII to associate with CD45. Therefore, Box A1-containing GII not only possesses a glucose-specific catalytic activity but also displays a mannose-specific lectin activity. This lectin activity is not of high enough affinity to allow GII to bind to branched mannose oligosaccharides alone because incubation of lysates from any one of the GII transfectants with mannan-agarose beads does not result in the binding GII to the beads (data not shown). From these data, we believe that both the active site and the lectin activity of Box A1 containing GII are required for the stable binding of GII to the N-linked carbohydrate on CD45.

The finding of lectin activity in addition to the enzymatic activity within a carbohydrate processing enzyme, though unusual, is not unprecedented. Both structural and biochemical evidence exists in the literature for this type of enzyme organization. The interplay between enzymatic activity and the lectin binding domain can be quite complex and unpredictable. For example, mutations in the ricin-like lectin motif of UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase do not affect the activity of the enzyme (31). However, in the case of cell surface 1,4-galactosyltransferase, the galactosyl transferase activity of depends on the ability of its lectin domain to bind its substrate, laminin (32). Further, the physical state of the enzyme can alter its substrate binding and affect enzymatic activity yet maintain its lectin binding capabilities. This is the case for -galactoside 2,6-sialyltransferase where dimerization reduces donor substrate binding but does not affect galactose binding (33). Therefore, it is conceivable that by utilizing both the GII active site and the lectin-binding domain, there may be modulation of either one or both of the activities causing the stable association of GII with CD45. For example, simultaneous engagement of the active site and the lectin domain may result in a stable association.

It is reasonable to deduce that the association between CD45 and GII may have significant biological implications for CD45. GII may act as a molecular chaperone that could help to retain CD45 within the ER longer than other proteins to allow for further post-translational modifications, the interaction with additional proteins, or regulated cell surface expression of CD45. The association with GII may also affect the enzymatic activity of CD45. While in the ER, reduced phosphatase activity could prevent the inappropriate dephosphorylation of proteins. In other compartments reduced activity may alter the signaling thresholds for cellular functions. As well, the stable interaction of GII with CD45 could perhaps lead to additional modifications on the carbohydrate of CD45, which could regulate CD45 function. Determining where the association between CD45 and GII occurs will be of interest. Our preliminary data suggest that the association does not take place on the cell surface, and because GII is an ER protein, we believe the interaction is at least initiated within the ER, thus supporting a function related to ER localization.

To date, most of the potential ligands and binding proteins of CD45 have been shown to interact with the carbohydrate on CD45, the biological significance of which is elusive. Our data suggest that GII is another example of a lectin that binds CD45. It is possible that CD45 does not have just one single ligand; rather there are multiple ligands that bind to CD45 carbohydrate and regulate the function of CD45.

We have begun to understand the biochemical basis for the GII-CD45 association, and this information has provided us with insight into the possible biological functions related to the interaction. These biological functions may help to identify the role that the carbohydrate and extracellular region CD45 plays in lymphocyte biology. As well, by understanding how GII interacts with its substrates, we hope to gain information into the role GII plays in the quality control system within the ER.

	ACKNOWLEDGEMENTS

We thank Dr. Ian Trowbridge for providing the PHA^r2.7 cell line and I3/2 hybridoma. We also thank Dr. Kevin Kane for critical review of this manuscript, Dr. Christopher Arendt for many helpful discussions, and Dr. Stuart Edmonds for the generation of the antiserum specific for the cytoplasmic domain of CD45.

	FOOTNOTES

^* This work was supported by the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Senior Scholar of the Alberta Heritage Foundation for Medical Research. To whom correspondence should be addressed. Tel.: 780-492-7710; Fax: 780-492-7521; E-mail: hanne.ostergaard@ualberta.ca.

Published, JBC Papers in Press, July 31, 2000, DOI 10.1074/jbc.M003088200

² T. A. Baldwin and H. L. Ostergaard, unpublished results..

	ABBREVIATIONS

The abbreviations used are: GII, glucosidase II; dNM, deoxynorjirmycin; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum; Endo F, endoglycosidase F.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				T. A. Baldwin and H. L. Ostergaard Developmentally Regulated Changes in Glucosidase II Association with, and Carbohydrate Content of, the Protein Tyrosine Phosphatase CD45 J. Immunol., October 1, 2001; 167(7): 3829 - 3835. [Abstract] [Full Text] [PDF]

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