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. 2012 Feb 27;22(7):983–996. doi: 10.1093/glycob/cws058

The glycan-binding properties of the cation-independent mannose 6-phosphate receptor are evolutionary conserved in vertebrates

Alicia C Castonguay 2, Yi Lasanajak 3, Xuezheng Song 3, Linda J Olson 2, Richard D Cummings 3, David F Smith 3,1, Nancy M Dahms 2,1
PMCID: PMC3355666  PMID: 22369936

Abstract

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) plays an essential role in the biogenesis of lysosomes by delivering newly synthesized lysosomal enzymes from the trans Golgi network to the endosomal system. The CI-MPR is expressed in most eukaryotes, with Saccharomyces cerevisiae and Caenorhabditis elegans being notable exceptions. Although the repertoire of glycans recognized by the bovine receptor has been studied extensively, little is known concerning the ligand-binding properties of the CI-MPR from non-mammalian species. To assess the evolutionary conservation of the CI-MPR, surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were carried out to probe the glycan-binding specificity of the Danio rerio CI-MPR. The results demonstrate that the D. rerio CI-MPR harbors three glycan-binding sites that, like the bovine CI-MPR, map to domains 3, 5 and 9 of its 15-domain-containing extracytoplasmic region. Analyses on a phosphorylated glycan microarray further demonstrated the unique binding properties of each of the three sites and showed that, similar to the bovine CI-MPR, only domain 5 of the D. rerio CI-MPR is capable of recognizing Man-P-GlcNAc-containing glycans.

Keywords: glycans, lectin, lysosome, receptor

Introduction

Lysosomes are acidified organelles containing a repertoire of over 60 different acid hydrolases that carry out the degradative metabolism of cellular macromolecules (Holtzman 1989). The lysosomal delivery of the acid hydrolases involves their recognition by mannose 6-phosphate receptors (MPRs), which comprise the ∼300-kDa cation-independent MPR (CI-MPR) and the ∼46-kDa cation-dependent MPR (CD-MPR). The MPRs play an essential role in lysosome biogenesis by binding to the mannose 6-phosphate (Man-6-P) tag on N-glycans of newly synthesized acid hydrolases and targeting these enzymes to lysosomes (Figure 1; Ghosh et al. 2003; Braulke and Bonifacino 2009). This phosphomannosyl recognition system is critical for normal development; deficiencies in GlcNAc-phosphotransferase (UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase; EC 2.7.8.17), the enzyme that carries out this post-translational modification of N-glycans, result in the lysosomal storage disorders mucolipidosis II and III (ML-II and ML-III; for reviews, see Kornfeld and Sly 2001; Cathey et al. 2010). The extracellular region of the CI-MPR contains 15 homologous, contiguous domains known as “MRH” (Man-6-P Receptor Homology) domains due to their similar size (∼150 residues), conserved cysteine distributions and conservation of residues (Munro 2001). Unlike the homodimeric CD-MPR, which contains only one Man-6-P-binding site per polypeptide, the CI-MPR contains three distinct glycan-binding sites, which map to domains 3, 5 and 9 of the bovine receptor. Extensive structural and site-directed mutagenesis studies of the mammalian receptors have shown that these three sites use the same four essential residues (Gln, Arg, Glu and Tyr) for phosphomannosyl recognition (for reviews, see Dahms et al. 2008; Kim et al. 2009; Castonguay et al. 2011; Figure 1).

Fig. 1.

Fig. 1.

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Sequence alignment of domains 3, 5 and 9 of the bovine and zebrafish CI-MPRs shows the conservation of essential residues for phosphomannosyl binding. The secondary structure of domain 3 is shown as determined by the crystal structure of domains 1–3 of the bovine CI-MPR (Olson, Dahms et al. 2004; Olson, Yammani et al. 2004), with arrows representing β-strands. The four residues essential for Man-6-P binding (i.e. Gln, Arg, Glu and Tyr) are shaded in yellow and are conserved in both species. Cysteine residues are shaded in cyan (domain 5 is missing two cysteine residues which are shaded in gray). Y679 (shaded in dark gray), which is positionally conserved with one of the missing Cys residues and involved in phosphodiester binding, is also present in domain 5 of the D. rerio CI-MPR. Potential N-linked glycosylation sites are highlighted in red. (Below) A schematic diagram of the CI- and CD-MPRs, highlighted are the domains that bind Man-6-P (blue) or Man-P-GlcNAc (red). The chemical structures of Man-6-P (blue) and Man-P-GlcNAc (red) in the β-anomeric configurations are shown.

The CI- and CD-MPRs are expressed in mammals, non-mammalian vertebrates and some invertebrates. However, Saccharomyces cerevisiae and Caenorhabditis elegans are notable exceptions as the MPRs are not found in these eukaryotes. The CI-MPR may have evolved from the CD-MPR due to repeated gene duplication events, given that the similarity of the single MRH domain of the mammalian CD-MPR is 14–37% to each of the 15 MRH domains of the CI-MPR (Lobel et al. 1988). Studies in mammals, including mouse, human and bovine, have shown that these receptors are functionally conserved, but only a few studies have been performed to characterize the receptors in non-mammalian vertebrates and invertebrates (for review, see Nadimpalli and Amancha 2010). Drosophila melanogaster was recently shown to have a protein (lysosomal enzyme receptor protein, LERP) that contains five MRH domains and is partially homologous to the mammalian CI-MPR. Interestingly, LERP can rescue the missorting of lysosomal enzymes in MPR-deficient mammalian cells, but lacks the essential signature motif residues for Man-6-P binding and does not bind to phosphomannan, a multivalent Man-6-P containing ligand (Dennes et al. 2005). These findings raise questions concerning the evolutionary development of the MRH domains in the MPRs and their essential roles in lysosome biogenesis.

The zebrafish (Danio rerio), a small cyprinid teleost fish representing one of the earliest forms of non-mammalian vertebrates, possesses both the CI- and CD-MPRs (Nolan et al. 2006). No evidence has been found for duplicate copies of either zebrafish MPR (Nolan et al. 2006). To date, only the zebrafish CD-MPR has been studied and shown to exhibit glycan-binding properties similar to the mammalian CD-MPR, as evidenced by the ability of the zebrafish CD-MPR to bind to a phosphomannan-containing resin and rescue the missorting of lysosomal enzymes in MPR-deficient mouse embryonic fibroblasts (Koduru et al. 2006). A structure-based sequence alignment of domains 3, 5 and 9 of the zebrafish and bovine CI-MPRs reveals the positional conservation in the zebrafish of all four key residues essential for Man-6-P recognition in domains 3, 5 and 9 (Figure 1). Additionally, this alignment shows the conservation in the zebrafish CI-MPR of Y679 (a residue important for phosphodiester binding), as well as the absence of two cysteine residues in domain 5 that are known to form a disulfide bridge in the binding pocket of the bovine CD-MPR (Roberts et al. 1998) and domain 3 of the bovine CI-MPR (Olson, Dahms, et al. 2004; Olson, Yammani, et al. 2004). Although domains 3, 5 and 9 of the zebrafish CI-MPR contain the signature motif residues needed for Man-6-P binding, the role of these domains in carbohydrate recognition in the zebrafish CI-MPR remains unclear. Recently, a zebrafish model of ML-II was developed using a morpholino-based knockdown strategy to generate GlcNAc-phosphotransferase-deficient embryos (Flanagan-Steet et al. 2009). Unexpectedly, the acid α-glucosidase (GAA), a lysosomal enzyme known to acquire the Man-6-P tag in mammals, fails to undergo mannose phosphorylation in zebrafish (Flanagan-Steet et al. 2009). This evidence suggests that the zebrafish may utilize additional pathways to target lysosomal enzymes to lysosomal compartments. Thus, to explore the potential of zebrafish as a model organism for the study of ML-II and other lysosomal storage disorders, it is imperative to assess the role of the zebrafish CI-MPR in carbohydrate recognition.

To probe the glycan-binding properties and ligand specificity of the zebrafish CI-MPR, we generated truncated forms of the zebrafish CI-MPR encoding domains 1–3, 5 and 9 and used quantitative surface plasmon resonance (SPR) analyses with lysosomal enzymes, β-glucuronidase and GAA enriched in either phosphomonoester- or phosphodiester-containing glycans. In addition, we probed a phosphorylated glycan array containing purified high-mannose-type glycans with zebrafish MPR constructs to determine the glycan specificities of the zebrafish CI-MPR. Our findings show for the first time that the zebrafish CI-MPR contains three carbohydrate-binding sites, localized to domains 3, 5 and 9, which display a similar preference for phosphomonoester-containing glycans (i.e. domains 1–3 and 9) and phosphodiester-containing glycans (i.e. domain 5) as the bovine CI-MPR.

Results

Expression and purification of domains 1–3, 5 and 9 of the zebrafish CI-MPR

To investigate the glycan-binding properties of the zebrafish CI-MPR, truncated forms of this receptor comprising the predicted carbohydrate-binding sites (domains 1–3, 5 and 9) were generated and expressed in Pichia pastoris or Spodoptera frugiperda (Sf9) as His-tagged, secreted proteins. We have shown previously that P. pastoris and Sf9 are effective heterologous host systems for the production of functional, soluble forms of the bovine MPRs (Reddy et al. 2004; Bohnsack et al. 2009). Because biochemical (Hancock et al. 2002) and structural (Olson, Dahms et al. 2004; Olson, Yammani et al. 2004) studies show that domain 3 of the bovine CI-MPR requires the presence of domains 1 and 2 to form a high-affinity Man-6-P-binding site, domains 1–3 of the zebrafish CI-MPR were expressed for this study. The His-tagged proteins were purified from the culture medium using nickel-nitrilotriacetic acid (Ni-NTA) chromatography and gel filtration to isolate a single species. As assessed by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and enzymatic deglycosylation, the purified zebrafish domains 1–3, 5 and 9 contain N-glycans and migrate with an apparent molecular mass of 53, 25 and 22 kDa, respectively (data not shown).

Conservation of the Man-6-P-binding sites in the zebrafish CI-MPR

Previously, we have used human β-glucuronidase, a homotetrameric lysosomal enzyme containing a heterogeneous population of phosphorylated and non-phosphorylated N-linked oligosaccharides, to define the binding affinity of various truncated forms of the MPRs to this model endogenous ligand (Reddy et al. 2004; Chavez et al. 2007; Bohnsack et al. 2009). We performed SPR analyses to evaluate the ability of zebrafish domains 1–3, 5 and 9 to bind a lysosomal enzyme. The data demonstrate the presence of a high-affinity Man-6-P-binding site in zebrafish domains 1–3 (Kd = 90 ± 6 nM; Figure 2A and D) and 9 (Kd = 136 ± 13 nM; Figure 2C and F). These results are consistent with the low nanomolar affinity binding observed with bovine CI-MPR's domains 1–3 and 9 toward β-glucuronidase (Westlund et al. 1991; Hancock et al. 2002). In contrast, zebrafish domain 5 exhibited relatively weak micromolar affinity binding (Kd = 5 ± 2 µM) to β-glucuronidase (Figure 2B and E), which is comparable with the low micromolar affinity displayed by bovine domain 5 for β-glucuronidase (Reddy et al. 2004). Taken together, the results show, for the first time, the ability of the zebrafish CI-MPR to bind a lysosomal enzyme. Furthermore, the results demonstrate that like the bovine receptor, the zebrafish CI-MPR contains two high-affinity and one low-affinity glycan-binding sites.

Fig. 2.

Fig. 2.

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SPR analysis of zebrafish domains 1–3, 5 and 9 binding to a lysosomal enzyme, β-glucuronidase. Increasing concentrations of β-glucuronidase (1, 2, 5, 10, 20, 40, 80 and 120 nM) were injected in a volume of 80 μL over the zebrafish domains 1–3 and 9 and reference flow cells at a rate of 40 μL/min. After 2 min, the solutions were replaced with buffer, and the complexes were allowed to dissociate for 2 min. Due to the limited availability of β-glucuronidase, β-glucuronidase was immobilized on a flow cell and domain 5 (1, 2, 5, 10 and 20 μM) was flowed over the sensor surface. Shown are representative sensorgrams at 40, 80, and 120 nM for β-glucuronidase on (A) zebrafish domains 1-3 and (C) domain 9 surfaces and for (B) domain 5 (5, 10, and 20 µM) on the β-glucuronidase surface. (D–F) The response at equilibrium (Req) was plotted vs the concentration β-glucuronidase and fitted by non-linear regression (SigmaPlot version 10.0) to determine equilibrium constants.

Determination of the glycan-binding properties of the zebrafish CI-MPR

Lysosomal enzymes become high-affinity ligands for the MPRs via the action of two enzymes during their transit through the secretory pathway. In early Golgi compartments, GlcNAc-phosphotransferase attaches GlcNAc-1-phosphate to the C-6 hydroxyl of one or more mannose residues to form a phosphodiester (Man-P-GlcNAc) on the N-glycans of lysosomal enzymes. A second enzyme, N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase (uncovering enzyme; EC 3.1.4.45), removes the GlcNAc moiety in the trans Golgi network to generate a phosphomonoester, Man-6-P. The phosphorylated N-glycans generated are heterogeneous in structure, differing in their size, number of phosphorylated mannose residues, presence of Man-6-P and/or Man-P-GlcNAc and the location of the phosphomannosyl residues on the different branches of the N-glycan, thus providing a diverse population of ligands presented to the MPRs (Varki and Kornfeld 1980, 1983).

To date, an assessment of the glycan-binding specificities of the individual carbohydrate-binding sites of the CI-MPR has only been carried out with the bovine receptor. Analyses utilizing glycan microarrays or lysosomal enzymes enriched in phosphorylated glycans have shown that the glycan-binding sites of the bovine CI-MPR differ from each other in their ability to recognize phosphomonoester- versus phosphodiester-containing glycans (Chavez et al. 2007; Bohnsack et al. 2009; Song et al. 2009). To determine the phosphomannosyl-binding preference of domains 1–3, 5 and 9 of the zebrafish CI-MPR, SPR analyses were performed using human GAA, a lysosomal enzyme, which has been enzymatically modified to contain only phosphomonoesters (GAA monoester) or phosphodiesters (GAA diester) (Chavez et al. 2007). Zebrafish domains 1–3 demonstrate a significant preference for the GAA monoester (Kd = 77 ± 1 nM), with no detectable binding observed for the GAA diester (Figure 3A and D). Similarly, zebrafish domain 9 exhibits a marked preference for the GAA monoester (Kd = 21 ± 2 nM), with minimal binding to the GAA diester surface (Figure 3C and F). In contrast, zebrafish domain 5 bound only to the phosphodiester surface (412 ± 50 nM; Figure 3B and E). To complement the above studies in which the receptor was immobilized on the sensor surface, similar amounts of GAA monoester or GAA diester were coupled to individual flow cells and increasing concentrations of zebrafish domains 1–3, 5 and 9 were flowed separately over the sensor surface. Zebrafish domains 1–3 interacted non-specifically with the sensor surface, similar to bovine domains 1–3 as described previously (Bohnsack et al. 2009; data not shown). Consistent with the above studies, zebrafish domain 5 exhibited a significant preference for the GAA diester (Kd = 4 ± 1 µM; GAA monoester, Kd = 16 ± 8 µM; Figure 4A and C), whereas zebrafish domain 9 interacted specifically only with the phosphomonoester surface (Kd = 39 ± 10 nM; Figure 4B and D).

Fig. 3.

Fig. 3.

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SPR analysis of GAA phosphomonoester or GAA phosphodiester lysosomal enzymes binding to immobilized zebrafish constructs. Similar amounts of purified zebrafish domains 1–3 (2857 RU), 5 (3008 RU) and 9 (2779 RU) were immobilized (separate flow cells) on a CM5 sensor chip. GAA phosphomonoester or GAA phosphodiester were injected in a volume of 80 µL over the zebrafish domains 1–3, 5 and 9 and reference flow cells at a rate of 40 µL/min. After 2 min, the solutions were replaced with buffer, and the complexes were allowed to dissociate for 2 min. Shown are representative sensorgrams for zebrafish (A) domains 1–3, (B) domain 5 and (C) domain 9 at 80, 120 and 250 nM of GAA (for zebrafish domains 1–3 and 9) and 40, 80 and 250 nM of GAA (zebrafish domain 5), comparing the response for GAA phosphomonoester (blue lines) and GAA phosphodiester (red lines). (D-F) The response at equilibrium (Req) was plotted versus the concentration (1, 2, 5, 10, 20, 40, 80, 120 and 250 nM) of GAA phosphomonoester or GAA phosphodiester (1, 2, 5, 10, 20, 40, 80, 120, 250 and 500 nM) and fitted by non-linear regression to a one-site saturation-binding model using the equation y = (Rmax × [MPR])/(Kd + [MPR]) (SigmaPlot version 10.0, Systat Software, Inc.).

Fig. 4.

Fig. 4.

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SPR analysis of zebrafish domains 5 and 9 binding to immobilized GAA phosphomonoester and GAA phosphodiester. Similar amounts of GAA phosphomonoester (4750 RU) or GAA phosphodiester (4370 RU) were immobilized (separate flow cells) on a CM5 sensor chip. Zebrafish domain 5 or 9 was injected in a volume of 80 µL over the sensor surface and reference flow cells at a rate of 40 µL/min. After 2 min, the solutions were replaced with buffer, and the complexes were allowed to dissociate for 2 min. Shown are representative sensorgrams for (A) domain 5 at 5, 10 and 20 µM and (B) domain 9 at 40, 80 and 120 nM comparing the response on GAA phosphodiester (red) and GAA phosphomonoester (blue) surfaces. (C and D) The response at equilibrium (Req) was plotted vs the concentration (0.5, 1, 5, 10 and 20 µM) of zebrafish domain 5 or 9 (5, 10, 20, 40, 80 and 120 nM) and fitted by non-linear regression to a one-site saturation-binding model using the equation y = (Rmax × [MPR])/(Kd + [MPR]) (SigmaPlot version 10.0, Systat Software, Inc.).

To directly measure the binding affinity of the zebrafish constructs to Man-6-P, inhibition studies were performed in which the GAA monoester was passed over the sensor surface containing either immobilized zebrafish domains 1–3 or domain 9 in the presence of increasing concentrations of Man-6-P or glucose 6-phosphate (Glc-6-P). Previous studies have utilized Glc-6-P as a control since it binds with low affinity (Kd = 1 − 8 × 10–2 M) to the full-length CD- and CI-MPRs, due to the equatorial orientation of the C-2 hydroxyl group of Glc-6-P (Tong and Kornfeld 1989). The binding affinity of zebrafish domain 5 to the GAA diester was not tested due to the limited availability of the competing ligand, GlcNAc-P-methyl mannoside. The results demonstrate that Man-6-P inhibited the interaction of zebrafish domains 1–3 (Ki = 280 µM; Figure 5A) and zebrafish domain 9 (Ki = 520 µM; Figure 5B) with the GAA monoester ligand. No appreciable inhibition was observed at the highest concentration of Glc-6-P (10 mM) for either zebrafish domains 1–3 (Figure 5A) or domain 9 (Figure 5B). Taken together, these studies demonstrate that the glycan-binding domains of the zebrafish CI-MPR exhibit distinct preferences for phosphomonoesters (domains 1–3 and 9) or phosphodiesters (domain 5) similar to the bovine CI-MPR.

Fig. 5.

Fig. 5.

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Inhibition of zebrafish domains 1–3 and 9 binding to GAA phosphomonoester by Man-6-P and Glc-6-P. Aliquots of GAA monoester (20 nM) were incubated with increasing concentrations of Glc-6-P or Man-6-P and injected over a flow cell coupled with (A) zebrafish domains 1–3 or (B) zebrafish domain 9. The response at equilibrium (Req) from the resulting sensorgrams is plotted as a percent of the maximal response against the log of the inhibitor concentration. The Ki was determined using non-linear regression (SigmaPlot, version 10.0).

Phosphorylated glycan microarray analyses reveal differences in glycan specificity

Our previous studies utilized a phosphomannosyl-containing glycan microarray, which contained a panel of non-phosphorylated and phosphorylated high-mannose-type glycans, to determine the glycan specificity of each of the bovine CI-MPR glycan-binding domains (Song et al. 2009). A new version of the phosphorylated glycan microarray was printed as previously described (Song et al. 2009) using AEAB (2-amino-N-(2-aminoethyl)-benzamide) derivatives of the glycans shown in Figure 6 for a similar analysis of zebrafish domains 1–3, 5 and 9 of the CI-MPR. Figure 7A shows the interrogation of this array with biotinylated concanavalin A (Con A), a plant lectin that binds all high-mannose-type N-glycans (glycans #1–21), as well as complex-type biantennary N-glycans (Baenziger and Fiete 1979; Brenckle and Kornfeld 1980; Brewer and Bhattacharyya 1986; glycans #23–25), but exhibits no detectable binding, as expected to LNnT-AEAB (glycan #22) or the biotin control (position #26; Figure 6). The nearly equivalent levels of Con A binding to the high-mannose- and complex-type biantennary N-glycans indicate that there were equivalent amounts of glycan printed at each position of the array. The positive signal for biotin detected with the labeled streptavidin is a control used for aligning the grid for analysis. This microarray was interrogated with the bovine CD- and CI-MPRs. Consistent with our previous studies using a similar phosphorylated glycan microarray (Song et al. 2009), the results show that the CI-MPR recognizes both phosphomonoester- and phosphodiester-containing glycans, whereas the CD-MPR preferentially binds phosphomonoester-containing glycans (Figure 7B and C). Taken together, these results validate the integrity of the glycans printed on the array and permit comparisons to the zebrafish constructs.

Fig. 6.

Fig. 6.

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Schematic representation of glycan structures present on the phosphorylated glycan array. Glycans #1–8 are the non-phosphorylated high-mannose-type oligosaccharides, glycans #9–15 are the phosphodiesters of high-mannose-type oligosaccharides, glycans #16–21 are the phosphomonoesters of high mannose-type oligosaccharides and glycans #23–25 are the complex-type biantennary oligosaccharides. Glycans #12 and #18 contain a mixture of two species. Mannose (green circles), N-acetylglucosamine (blue squares), glucose (blue circles), galactose (yellow circles), N-acetylneuramic acid (purple diamond) and phosphate (P). α- and β-linkages are denoted by a and b, respectively, as in a3 or b4.

Fig. 7.

Fig. 7.

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The phosphorylated glycan microarray. The microarray was printed as described in the text and the structures of the individual glycans are identified by their glycan number as indicated in Figure 6. Glycans #1–8 are the high-mannose-type glycans, glycans #9–15 the phosphodiesters of high-mannose-type glycans and glycans #16–21 the phosphomonoesters of high-mannose-type glycans. Glycans #22–25 are control glycans LNnT, NA2, α2-3-sialylated-NA2 and α2-6-sialylated-NA2, respectively. (A) Biotinylated Con A (1 µg/mL) binding detected with 0.5 µg/mL of Cy5-labeled streptavidin; (B) The bovine CD-MPRHis (0.1 µg/mL) detected with anti-Penta His antibody (5 µg/mL) and Alexa633-labeled Goat anti-mouse IgG (5 µg/mL). (C) The bovine CI-MPR (0.05 µg/mL) detected with rabbit antibody B14.5 (1:250) and Cy5-labeled goat anti-rabbit IgG (5 µg/mL) Alexa633-labeled Goat anti-mouse IgG (5 µg/mL). Zebrafish CI-MPR's domains 1–3 (D), domain 5 (E) and domain 9 (F) were assayed at 10, 50 and 1 µg/mL, respectively, as described in the text. The data in (D)–(F) are shown for glycan derivatives that correspond to glycans #9–25 (Figure 6). Concentration-dependent analyses of zebrafish CI-MPR's domains 1–3, 5 and 9 are summarized in Supplementary data, Figures S1–S6. The analyses were single experiments where each glycan is printed in replicates of six, and the relative fluorescence unit (RFU) values are the mean of four remaining values after removing the highest and lowest signals from each set of replicates. The error bars are the standard deviation.

The interaction of the His-tagged zebrafish CI-MPR constructs with the phosphorylated glycan microarray was detected using an anti-His antibody for Dom 1–3 and 5, whereas Dom 9 was detected with polyclonal antibody 2563. No appreciable binding by the zebrafish constructs was detected to the non-phosphorylated high-mannose glycans (glycans #1–8), which correlates with previous studies showing that disaccharides and trisaccharides are poor inhibitors for the CI-MPR (Distler et al. 1991; Tomoda et al. 1991). Like bovine domains 1–3, 5 and 9 (Bohnsack et al. 2009), the zebrafish domains 1–3, 5 and 9 interact specifically with the phosphorylated glycans (Figure 7D–F). Consistent with the SPR data, the analysis of zebrafish domains 1–3 (Figure 7D) and 9 (Figure 7F) on the phosphorylated glycan array shows that these domains are highly specific for phosphomonoesters. In contrast, zebrafish domain 5 recognizes phosphodiesters but exhibits little or no binding toward phosphomonoester-containing glycans (Figure 7E). To determine the relative order of binding of the various phosphorylated glycans to the zebrafish domains of the CI-MPR, each domain was subjected to a concentration-dependent analysis (Supplementary data, Figures S1–S6), and the domain concentrations at half maximal binding to each glycan are summarized in Table I. Zebrafish domain 5 binds all phosphodiester-containing glycans with similar strength (estimated Kd = 0.6–3 μM), with the exception of glycan #11. As observed with domain 5 of the bovine CI-MPR (Bohnsack et al. 2009), this region of the receptor does not recognize a Man7 isomer containing a single Man-P-GlcNAc on arm B and/or C (Figures 6 and 8). Although zebrafish domains 1–3 and 9 are specific for phosphomonoester-containing glycans, neither construct is capable of recognizing Man8 and Man9 glycans with a single phosphomonoester on arm C (glycans #19 and #20). Zebrafish domains 1–3 and 9 recognize all the remaining Man-6-P-containing glycans with high affinity, with less variability in affinity observed for domain 9 (i.e. Kd ranges from 56 to 540 nM for domains 1–3 versus 18–45 nM for domain 9).

Table I.

Estimated, relative binding strengths of zebrafish CI-MPR domains based on half-maximal binding to the phosphorylated glycan microarray compared with the binding affinities toward lysosomal enzymes

Glycan # Designation Dom 1–3-His
 Dom 5-His
 Dom 9-His
1/2 maximum binding (µg/mL)a Estimated Kda 1/2 maximum binding (µg/mL) Estimated Kd 1/2 maximum binding (µg/mL) Estimated Kd
9 GPM6 >500 NA 36 1400 DNB NA
10 2(GP)M6 >200 NA 43 1700 DNB NA
11 GPM7(2) DNB NA DNB NA DNB NA
12 2(GP)M7(1)+(3) >200 NA 16 640 DNB NA
13 GPM8(1) >500 NA 63 2500 DNB NA
14 GPM9 >500 NA 76 3000 DNB NA
15 2(GP)M9 >200 NA 23 920 DNB NA
16 PM6 30 540 >1000 NA 1.00 45
17 2(P)M6 3.1 56 >500 NA 0.42 19
18 2(P)M7(1)+(3) 4.4 80 >500 NA 0.40 18
19 PM8(1) >200 NA DNB NA DNB NA
20 PM9 >200 NA DNB NA DNB NA
21 2(P)M9 48 870 DNB NA 0.80 36
Lysosomal enzyme Dom 1–3-His (Kd)b Dom 5-His (Kd)b Dom 9-His (Kd)b
GAA diester DNB 412 (4000) DNB
GAA monoester 77 (16,000) 21 (39)
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NA, not applicable; DNB, did not bind.

aThe half maximal binding concentration (µg/mL) for each MPR construct was estimated from graphs of the data shown in Supplementary data, Figure S1. The estimated Kds are shown in nM concentrations using the following molecular weight assignments: Dom 1–3-His, 55,000 Da; Dom 5-His, 25,000 Da; Dom 9-His, 22,000 Da.

bKd (nM) were obtained from SPR analyses shown in Figure 3. Values in parentheses were obtained from SPR analyses shown in Figure 4.

Fig. 8.

Fig. 8.

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Glycan specificity of the zebrafish CI-MPR. Phosphorylated glycan structures recognized by the zebrafish constructs as determined by the glycan microarray analyses are shown. Domains 1–3 and 9 bind phosphomonoester-containing glycans (blue bar), whereas domain 5 displays a preference for phosphodiester-containing glycans (red bar). Relative binding affinities are denoted by shading, with light red and light blue representing a lower binding affinity (Table I). The absence of a bar denotes no significant binding above background. In contrast to the zebrafish CI-MPR, the bovine CI-MPR's domain 9 binds with high affinity to glycans #19 and #20 (blue stars; Bohnsack et al. 2009). The inset shows the structure of the glycan transferred to Asn during N-glycosylation following the removal of three glucose residues by ER glucosidases I and II. The three arms of the N-glycan are labeled and their mannose residues are designated A–I as shown. Mannose (green circles), N-acetylglucosamine (blue squares) and phosphate (P).

Discussion

The MPRs play an essential role in lysosome biogenesis by targeting phosphomannosyl-containing acid hydrolases to endosomal compartments, thus providing lysosomes with a repertoire of over 60 different soluble acid hydrolases to carry out various degradative processes. Studies in mammals, including mouse, bovine and human, have shown the functional conservation of the MPRs, but only a few studies have been performed to characterize the receptors in non-mammalian vertebrates. Previous studies in zebrafish have shown the presence of both the MPRs (Nolan et al. 2006), but the zebrafish CD-MPR has been only partially characterized in that it was shown to bind phosphomannan Sepharose, which is a property shared with the mammalian CD-MPR (Koduru et al. 2006). In the current study, the binding properties and ligand specificities of the zebrafish CI-MPR carbohydrate-binding domains (domains 1–3, 5 and 9) were biochemically determined using SPR and a phosphorylated glycan array, and the results reveal interesting relationships between the zebrafish and mammalian CI-MPRs.

Our previous crystal structures of the bovine CD-MPR (Roberts et al. 1998; Olson et al. 1999) and domains 1–3 of the bovine CI-MPR (Olson, Dahms et al. 2004; Olson, Yammani et al. 2004) revealed key conserved residues (Gln, Arg, Glu and Tyr) that serve as a signature motif for Man-6-P binding, which bind the 2-, 3- and 4-hydroxyl groups of the mannose ring in a similar manner. A structure-based sequence alignment of domains 3, 5 and 9 of the zebrafish and bovine CI-MPRs demonstrated the positional conservation of all four key residues (Gln, Arg, Glu and Tyr) essential for Man-6-P recognition in domains 3, 5 and 9 of the zebrafish CI-MPR (Figure 1). Additionally, this alignment showed the conservation of Y679, a residue involved in Man-P-GlcNAc recognition by domain 5. Based on this alignment, we hypothesized that domains 1–3, 5 and 9 of the zebrafish would exhibit similar glycan-binding properties as the bovine CI-MPR. To test this hypothesis, truncated soluble forms of the zebrafish CI-MPR containing domains 1–3, 5 or 9 alone were assayed by SPR for their ability to bind β-glucuronidase, a lysosomal enzyme containing heterogeneous N-glycans. Similar to the nanomolar affinity observed for bovine domains 1–3 (Kd = 1 ± 0.5 nM) and 9 (Kd = 70 ± 8 nM; Bohnsack et al. 2009), the zebrafish domains 1–3 and 9 exhibited binding affinities for β-glucuronidase in the nanomolar range (Kd = 90 ± 6 nM and Kd = 136 ± 13 nM, respectively; Figure 2A and C). Zebrafish domain 5, consistent with bovine domain 5 (Bohnsack et al. 2009), binds β-glucuronidase with a much lower affinity (Kd = 5 ± 2 µM) than bovine domains 1–3 and 9. Taken together, these results show the conservation of a glycan-binding site in domains 1–3, 5 and 9 of the zebrafish CI-MPR and the ability of these domains to recognize a lysosomal enzyme.

Previously, we have shown that bovine domains 1–3 and 9 bind with nanomolar affinity to phosphomonoesters, whereas domain 5 preferentially binds phosphodiesters with micromolar affinity (Bohnsack et al. 2009). Similar to the preferences displayed by the glycan recognition sites of the bovine CI-MPR, the zebrafish domains 1–3 and 9 bound phosphomonoesters (Kd = 77 ± 1 nM, Kd = 21 ± 2 nM, respectively) with little to no binding detected to the phosphodiester (Figures 3 and 4). Furthermore, inhibition studies using Man-6-P exhibited the high degree of specificity of zebrafish domains 1–3 and 9 for the phosphomonoester Man-6-P, with no detectable inhibition observed with Glc-6-P (Figure 5). Zebrafish domain 5 exhibited preferential binding to the phosphodiester (Kd = 412 ± 50 nM), similar to the results obtained for bovine domain 5 (Kd = 720 ± 150 nM). These findings demonstrate the conserved preference for phosphomonoesters by domains 1–3 and 9 and phosphodiesters by domain 5 of the zebrafish CI-MPR. Although the signature motif residues (Glu, Gln, Tyr and Arg) are critical for glycan binding, as they serve as hydrogen-bonding partners for the hydroxyl groups of mannose, other residues present in the glycan-binding domains are also important for carbohydrate recognition. Our crystal structure of domains 1–3 has revealed the importance of S386 (Olson, Dahms et al. 2004; Olson, Yammani et al. 2004), a residue that contacts the phosphate moiety of Man-6-P, and is conserved in zebrafish domain 3 (Figure 1). Additionally, structural determination and mutagenesis studies of the bovine domain 5 have identified Y679 as important in phosphodiester recognition (Olson et al. 2010). Given that zebrafish domains 1–3, 5 and 9 possess all the residues involved in carbohydrate recognition and conserved structural elements (e.g. the presence or the absence of disulfide bonds), the above results suggest that the zebrafish domains 1–3, 5 and 9 utilize these conserved residues for carbohydrate recognition and that these domains adopt the conserved MRH fold.

Recently, we developed a phosphorylated glycan microarray to define the glycan-binding specificities of the three phosphomannosyl recognition sites in the bovine CI-MPR. The results demonstrated that each of the carbohydrate recognition sites of the CI-MPR exhibits a unique profile of preferred glycans, and together, the three carbohydrate-binding sites in the full-length CI-MPR account for the broad range of phosphorylated N-glycans recognized by the CI-MPR (Figure 7C and Bohnsack et al. 2009). This unique profile is partially maintained in the zebrafish CI-MPR (Figure 8): zebrafish domain 5 prefers phosphodiester-containing glycans; zebrafish domains 1–3 and 9 prefer phosphomonoester-containing glycans, but neither interacts with glycans PM8(1) (glycan #19) and PM9 (glycan #20), both of which have a single phosphate on the terminal α1,2-linked mannose of the C arm. In contrast, domain 9 of the bovine CI-MPR bound the phosphomonoester-containing glycans #16–21 with a similar high affinity, ranging from 26 to 66 nM (Bohnsack et al. 2009). Taken together, these results indicate that the full-length zebrafish CI-MPR recognizes a more limited range of phosphorylated glycans than the bovine CI-MPR. In addition, these results also predict that the structure of CI-MPR's domain 9 must differ between the bovine and the zebrafish to account for the observed disparity in the recognition of glycans #19 and #20.

Collectively, the above results suggest that the gene duplication events hypothesized to give rise to the CI-MPR occurred prior to the evolution of the zebrafish. The similar range of phosphorylated glycans recognized by the zebrafish and bovine CI-MPRs indicate the conservation of the Man-6-P recognition system in non-mammalian vertebrates. However, the inability of the zebrafish CI-MPR, unlike the bovine CI-MPR, to recognize glycans PM8(1) and PM9 which bear a single phosphate on the terminal mannose of arm C may reflect the type of N-glycan structures that are acquired by zebrafish lysosomal enzymes in vivo, which are predominantly biantennary high-mannose type or complex-type in nature (Takemoto et al. 2005). Further support of the conservation of the glycan-binding properties of the CI-MPR and its essential role in trafficking lysosomal enzymes has come from a recent study in chickens. The siRNA knockdown of the CI-MPR in chicken embryonic fibroblasts resulted in the missorting and secretion of lysosomal enzymes into the medium and impaired the endocytosis of Man-6-P containing ligands at the cell surface (Yadavalli and Nadimpalli 2010). However, additional studies on the MPRs from other non-mammalian vertebrates and invertebrates are needed to identify a clear path in the evolution of the MRH domain and the unique ability of the MPRs to bind phosphorylated glycans.

Materials and methods

Generation of constructs

Zebrafish CI-MPR cDNAs (National Center for Biotechnology Information access number: NM_001039627) encoding domains 1–3 (residues 1–426), 5 (residues 581–725) and 9 (residues 1176–1317) were codon optimized and synthesized by GenScript Corp. (New Jersey). In addition, the constructs were engineered to contain a C-terminal tag of six histidine residues to facilitate purification. Zebrafish cDNA constructs (domains 5 and 9) were cloned (utilizing EcoRI and XbaI of the multiple cloning site) into the pPICZαA expression vector (Invitrogen, Grand Island, NY) containing the 89-residue yeast α-factor signal sequence at the N terminus for expression in P. pastoris following methanol induction. As a result of this strategy, the mature secreted protein contained four additional amino acids (EAEA) to the N terminus (Reddy et al. 2004). Zebrafish domain 1–3 cDNA construct was cloned into the pVL1392/93 baculovirus transfer vector (Pharmingen, San Diego, CA) and expressed in Spodoptera frugiperda (Sf9, Expression Systems, Woodland, CA) insect cells as described previously (Bohnsack et al. 2009).

Protein expression and purification

The zebrafish cDNA constructs were linearized with SacI or PmeI and transformed into P. pastoris wild-type strain X-33 (Invitrogen) by electroporation following the protocol supplied by Invitrogen. Zeocin-resistant clones were isolated as described previously (Reddy and Dahms 2002) and grown overnight at 28°C in minimal media [0.34% yeast nitrogen base without amino acids, 70 mM potassium phosphate (pH 6.0), 38 mM ammonium sulfate, 4 × 10–5% biotin and 2% glycerol]. To induce the expression of the recombinant protein, the cells were resuspended to an OD600 of 1 in minimal media in which glycerol was replaced with 0.5% methanol as the sole carbon source and then grown at 25°C. Cultures were supplemented with 0.5% methanol every 24 h and harvested after 3 days. Following the removal of cells by centrifugation, medium from insect cell or yeast cultures was concentrated by filtration using Amicon stirred cells and then dialyzed extensively against Ni-NTA-binding buffer containing 50 mM Tris (pH 8.0) and 300 mM NaCl. The dialyzed medium was passed over a Ni-NTA agarose (Qiagen, Valencia, CA) column, washed and then eluted with Ni-NTA-binding buffer containing 50–400 mM imidazole. Following affinity chromatography purification, all proteins were extensively dialyzed against buffer containing 25 mM imidazole, pH 6.5, 150 mM NaCl and concentrated by filtration using Vivaspin spin columns containing a polyethersulfone membrane with a 5-kDa nominal molecular mass limit (GE Healthcare, Piscataway, NJ). Concentrated proteins were subjected to gel filtration using a Superose 12 column (10 × 300 mm) to isolate a single species. The Bradford protein assay (Bio-Rad, Hercules, CA), with bovine serum albumin (BSA) as the standard, was used to estimate protein yields.

Endoglycosidase H digestion

Purified zebrafish constructs were incubated with endoglycosidase H in buffer containing 25 mM imidazole, pH 6.5, 150 mM NaCl at 37°C, and aliquots were removed at 0 h, 10 min, 30 min, 1 h and 16 h. The samples were resolved by SDS–PAGE and detected by Coomassie blue or silver staining as described by the manufacturer (Bio-Rad).

Generation of recombinant GAA with GlcNAc1-P-6-Man (GAA diester) and Man-6-P (GAA monoester)

The GAA diester and the GAA monoester were prepared from recombinant human GAA with high-mannose-type glycans using recombinant human GlcNAc-phosphotransferase, uncovering enzyme and/or sweet potato purple acid phosphatase in vitro as described by Chavez et al. (2007).

Purification of human β-glucuronidase

Human β-glucuronidase was collected from serum-free conditioned medium from cells that over-express this lysosomal enzyme (MTX 3.2 cells were generously provided by Dr William Sly, St. Louis University School of Medicine, St Louis, MO). β-Glucuronidase was purified by affinity chromatography on a CI-MPR Affigel-10 column as described previously (Marron-Terada et al. 1998).

SPR analysis

All SPR measurements were performed at 25°C using a BIAcore 3000 instrument (GE Healthcare). The zebrafish constructs were immobilized on a CM5 sensor chip (GE Healthcare) following the activation of the surface using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The proteins were injected onto the activated dextran surface at a concentration of 5–10 μg/mL in 10 mM sodium acetate buffer, pH 4.5 (Dom 1–3), pH 5.5 (Dom 5) or pH 5.0, using immobilization buffer (10 mM 2-(N-morpholinoethane)sulfonic acid (MES), pH 6.5, 150 mM NaCl and 0.005% (vol/vol) P20) as the running buffer. After coupling, unreacted N-hydroxysuccinimide ester groups were blocked with ethanolamine. The reference surface was treated in the same manner except that protein was omitted. To assess binding affinities, GAA monoester and GAA diester were prepared in running buffer (25 mM imidazole, pH 6.5, 150 mM NaCl) and were injected in a volume of 80 μL over the coupled and reference flow cells at a flow rate of 40 µL/min. After 2 min, the solutions containing the purified proteins were replaced with buffer and the complexes were allowed to dissociate for 3 min. The sensor chip surface was regenerated with a 10-µL injection of 10 mM Man-6-P at a flow rate of 10 µL/min. The surface was allowed to re-equilibrate in running buffer for 1 min prior to subsequent injections. The response at equilibrium (Req) for each concentration of protein was determined by averaging the response over a 10-s period within the steady state region of the sensorgram using the BIAevaluation software package (4.0.1). The response at equilibrium (Req) was plotted versus the concentration of the protein and fit by nonlinear regression to a one-site saturation-binding model using the equation y = (Rmax × [MPR])/(Kd + [MPR]) (SigmaPlot version 10.0 Systat Software, Inc.). For inhibition experiments, GAA monoester (20 nM) was flowed over the sensor surface immobilized with zebrafish domains 1–3 and 9 in the presence of increasing concentrations of Glc-6-P (Sigma) or Man-6-P (Sigma-Aldrich, St Louis, MO). The response at equilibrium (Req) for each concentration of protein was determined by averaging the response over a 10-s period within the steady-state region of the sensorgram using the BIAevaluation software package. The Req was plotted versus the concentration of inhibitor and was fit by non-linear regression to a one-site inhibition model (SigmaPlot version 10.0, Systat Software, Inc.). All response data were double or triple referenced (Myszka 2000), where controls for the contribution of the change in the refractive index were performed in parallel with flow cells derivatized in the absence of protein and subtracted from all binding sensorgrams.

Glycan microarray analyses

The glycan-AEAB derivatives of the high-mannose N-glycans and their phosphomonoesters and phosphodiesters were generated as described previously (Song et al. 2009). The current version of the phosphorylated glycan microarray is comprised of 21 glycans, which are defined in Figure 6. The previous phosphorylated glycan microarray (Song et al. 2009) was made up of 24 glycans, but one of the mono-phosphodiester derivatives of a Man7 isomer and two of the mono-phosphomonoester of Man7 isomers were no longer available. Aliquots (0.005 mL) of the glycan derivatives stored in deionized water were mixed with an equal volume of 200 mM sodium phosphate buffer (pH 8.5) to make up a final concentration of 100 µM glycan in 100 mM sodium phosphate buffer and printed onto N-hydroxysuccinimide-activated glass slides via their free alkylamine using a non-contact printer as described previously (Song et al. 2009). To analyze proteins bound to the glycan array, the slides were fitted with a 16-chamber adapter and subarrays were rehydrated in assay buffer (50 mM imidazole, pH 6.5, 150 mM NaCl and 10 mM MnCl2 for Con A and bovine CD-MPR or 25 mM imidazole, pH 6.5, 150 mM NaCl and 10 mM ethylenediaminetetraacetic acid (EDTA) for bovine or zebrafish CI-MPR's domains 1–3, 5 or 9. The slides were incubated with proteins in 70 µL of specified concentrations in appropriate buffers containing 1% BSA and 0.05% Tween-20. After 1 h at room temperature, the solutions were removed and the individual chambers were washed with buffer. Biotinylated Con A, bovine CD-MPR and bovine CI-MPR were detected as described previously (Song et al. 2009). The CI-MPR purified from bovine fetal serum (Tong and Kornfeld 1989) was detected with rabbit antibody B14.5 (1:250) followed by 5 µg/mL of Cy5-labeled goat anti-rabbit IgG (Abcam, Cambridge, MA). Bovine CD-MPRHis (Sun et al. 2005) and the His-tagged zebrafish domains 1–3 and 5 were detected with 5 µg/mL each of an anti-penta-His monoclonal antibody (Qiagen) and an Alexa633-labeled Goat anti-mouse IgG (Invitrogen). The zebrafish domain 9 was detected with polyclonal antibody 2563 diluted 1:250 followed by Cy5-labeled goat anti-rabbit IgG (Abcam, Cambridge, MA). To determine relative affinities of the zebrafish domains for their respective phosphorylated glycan ligands, the concentrations of the domains were varied in assays on the phosphorylated glycan microarray and the estimated concentrations of protein at half maximal binding to each glycan were compared. None of the secondary detection reagents bound to glycans on the array as determined by omitting MPRs or zebrafish MPR domains (data not shown).

Supplementary data

Supplementary data for this article is available online at http://glycob.oxfordjournals.org/.

Funding

This work was supported by the National Institutes of Health (DK042667 to N.M.D. and GM085448 to D.F.S.).

Conflict of interest

None declared.

Abbreviations

AEAB, 2-amino-N-(2-aminoethyl)-benzamide; BSA, bovine serum albumin; CD-MPR, cation-dependent mannose 6-phosphate receptor; CI-MPR, cation-independent mannose 6-phosphate receptor; Con A, concanavalin A; EDTA, ethylenediaminetetraacetic acid; GAA, acid α-glucosidase; GlcNAc-phosphotransferase, UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase; Glc-6-P, glucose 6-phosphate; LERP, lysosomal enzyme receptor protein; Man-6-P, mannose 6-phosphate; MES, 2-(N-Morpholino)ethanesulfonic acid; ML, mucolipidosis; MPR, mannose 6-phosphate receptor; MRH, mannose 6-phosphate receptor homology; Ni-NTA, nickel-nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SPR, surface plasmon resonance.

Supplementary Material

Supplementary Data
supp_22_7_983__index.html (1.7KB, html)

Acknowledgements

We thank Dr William M. Canfield for providing acid α-glucosidase (GAA) phosphomonoester and GAA phosphodiester, and Richard N. Bohnsack for assistance in SPR analyses. The BIAcore 3000 instrument (Protein and Nucleic Acid Core Facility, Medical College of Wisconsin) was purchased through a grant from the Advancing a Healthier Wisconsin program.

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Associated Data

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Supplementary Materials

Supplementary Data
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supp_cws058_cws058supp_fig3.tif (274KB, tif)
supp_cws058_cws058supp_fig4.tif (511.5KB, tif)
supp_cws058_cws058supp_fig5.tif (445.9KB, tif)
supp_cws058_cws058supp_fig6.tif (346.6KB, tif)

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