Domain 5 of the Cation-Independent Mannose 6-Phosphate Receptor Preferentially Binds Phosphodiesters (Mannose 6-Phosphate N-Acetylglucosamine Ester)

Abstract

The 300 kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46 kDa cation-dependent MPR (CD-MPR) are key components of the lysosomal enzyme targeting system that bind newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases and divert them from the secretory pathway. Previous studies have mapped two high-affinity Man-6-P binding sites of the CI-MPR to domains 1-3 and 9 and one low-affinity site to domain 5 within its 15-domain extracytoplasmic region. A structure-based sequence alignment predicts that domain 5 contains the four conserved residues (Gln, Arg, Glu, Tyr) identified as essential for Man-6-P binding by the CD-MPR and domains 1-3 and 9 of the CI-MPR. Here we show by surface plasmon resonance (SPR) analyses of constructs containing single amino acid substitutions that these conserved residues (Gln-644, Arg-687, Glu-709, Tyr-714) are critical for carbohydrate recognition by domain 5. Furthermore, the N-glycosylation site at position 711 of domain 5, which is predicted to be located near the binding pocket, has no influence on the carbohydrate binding affinity. Endogenous ligands for the MPRs that contain solely phosphomonoesters (Man-6-P) or phosphodiesters (mannose 6-phosphate N-acetylglucosamine ester, Man-P-GlcNAc) were generated by treating the lysosomal enzyme acid alpha-glucosidase (GAA) with recombinant GlcNAc-phosphotransferase and uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase). SPR analyses using these modified GAAs demonstrate that, unlike the CD-MPR or domain 9 of the CI-MPR, domain 5 exhibits a 14-18-fold higher affinity for Man-P-GlcNAc than Man-6-P, implicating this region of the receptor in targeting phosphodiester-containing lysosomal enzymes to the lysosome.

Structural Insights into the Mechanism of pH-dependent Ligand Binding and Release by the Cation-dependent Mannose 6-Phosphate Receptor

“…Furthermore, the previously reported low concentration of Mn 2ϩ in human serum (40 -120 nM), along with the measured binding affinity of the sCD-MPR for Mn 2ϩ (K d ϭ 600 M) (10), is consistent with the receptor lacking bound Mn 2ϩ at the cell surface. The observation that Asp-103 and His-105 are not conserved in the three Man-6-P binding pockets of the homologous CI-MPR (32,33), an MPR that does not display either cation dependence or loss of ligand binding activity at pH 7.4 (6, 7), further supports a role for these residues in modulating ligand binding by the CD-MPR at the cell surface. Our finding that the receptor is bound to a non-phosphorylated oligosaccharide at pH 7.4 suggests a role for the CD-MPR at the cell surface in binding proteins containing high mannose-type glycans, a result consistent with a previous study that showed that the non-phosphorylated ␣1,2-linked dimannoside was only a 14-fold weaker inhibitor of lysosomal enzyme binding to the receptor than Man-6-P (25).…”

“…5, A and B). Four residues of the binding pocket (Gln-66, Arg-111, Glu-133, and Tyr-143), which interact with the 2-, 3-, and 4-hydroxyl groups of the mannose ring, are conserved in the CD-MPR and the three distinct Man-6-P binding sites of the CI-MPR, and mutagenesis studies have confirmed their critical role in carbohydrate recognition (10,27,33,35). The reorganization of the binding pocket in the absence of ligand, particularly the relocation of loop D, functions to maintain the correct positioning of three (Arg-111, Gln-66, and Tyr-143) out of these four residues known to be essential for Man-6-P binding in the absence of ligand (Fig.…”

Biochemical and pharmacological characterization of different recombinant acid α-glucosidase preparations evaluated for the treatment of Pompe disease

“…Methods for the purification of the transgenically-produced rhGAA (tgGAA), the CHO-GAA, and the carbohydrate engineered form of rhGAA (HP-GAA) are described elsewhere [6,18,19]. The purity of the various forms of GAA used in these studies was ≥95% as judged by SDS gel electrophoresis.…”

Glycan Microarray Analysis of P-type Lectins Reveals Distinct Phosphomannose Glycan Recognition

“…The difficulty in defining the specific interactions of the P-type lectins with glycans containing Man-6-P or Man-P-GlcNAc has been due to the lack of defined glycan structures that can be used for direct binding or inhibition studies. Recently, domain 5 of the CI-MPR was shown to prefer Man-P-GlcNAc phosphodiesters, whereas domains 3 and 9 favored Man-6-P monoesters (22). These studies utilized recombinant ␣-glucosidase that was generated by treatment with recombinant GlcNAc-phosphotransferase alone or in combination with recombinant uncovering enzyme and binding was determined by surface plasmon resonance (22).…”

“…Recently, domain 5 of the CI-MPR was shown to prefer Man-P-GlcNAc phosphodiesters, whereas domains 3 and 9 favored Man-6-P monoesters (22). These studies utilized recombinant ␣-glucosidase that was generated by treatment with recombinant GlcNAc-phosphotransferase alone or in combination with recombinant uncovering enzyme and binding was determined by surface plasmon resonance (22). However, this study did not provide information about binding of the complete extracytoplasmic domains of both MPRs, nor did it identify the contributions of the underlying high-mannose structures or the influence of the particular mannose residues that were phosphorylated.…”