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

Xuezheng Song,Yi Lasanajak,Linda J Olson,Marielle Boonen,Nancy M Dahms,Stuart Kornfeld,Richard D Cummings,David F Smith

doi:10.1074/jbc.m109.056119

Abstract

The specificity of the cation-independent and -dependent mannose 6-phosphate receptors (CI-MPR and CD-MPR) for high mannose-type N-glycans of defined structure containing zero, one, or two Man-P-GlcNAc phosphodiester or Man-6-P phosphomonoester residues was determined by analysis on a phosphorylated glycan microarray. Amine-activated glycans were covalently printed on N-hydroxysuccinimide-activated glass slides and interrogated with different concentrations of recombinant CD-MPR or soluble CI-MPR. Neither receptor bound to non-phosphorylated glycans. The CD-MPR bound weakly or undetectably to the phosphodiester derivatives, but strongly to the phosphomonoester-containing glycans with the exception of a single Man7GlcNAc2-R isomer that contained a single Man-6-P residue. By contrast, the CI-MPR bound with high affinity to glycans containing either phospho-mono- or -diesters although, like the CD-MPR, it differentially recognized isomers of phosphorylated Man7GlcNAc2-R. This differential recognition of phosphorylated glycans by the CI- and CD-MPRs has implications for understanding the biosynthesis and targeting of lysosomal hydrolases.

Highlights

Ering these enzymes to the lysosome [1,2,3,4]
Because the recombinant CD-MPR was His-tagged, we explored its interaction with the microarray at 50 ␮g/ml using anti-His to detect binding, which has been successfully used in prior studies for His-tagged proteins in glycan microarray analyses [42]
The high mannose-type N-glycans of many lysosomal hydrolases are modified in the Golgi apparatus by the GlcNAc-phosphotransferase and the donor UDP-GlcNAc to contain various amounts of Man-P-GlcNAc phosphodiesters [8, 43]

Summary

Introduction

Ering these enzymes to the lysosome [1,2,3,4]. The key determinant for recognition of lysosomal hydrolases by the CI- and CDMPR is the presence of mannose 6-phosphate (Man-6-P) on select mannose residues of the high mannose-type N-glycans. Sorting out the binding specificities of the different domains of the MPRs would be greatly facilitated by an analysis of their affinity for N-glycan structures containing the various combinations of Man-P-GlcNAc and Man-6-P residues that can potentially be generated on lysosomal hydrolases in vivo.

Results

Conclusion