Identification of Residues Essential for Carbohydrate Recognition by the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

Michael K Hancock,Darin J Haskins,Guangjie Sun,Nancy M Dahms

doi:10.1074/jbc.m109855200

Michael K Hancock, Darin J Haskins + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m109855200

Copy DOI

Abstract

Two distinct mannose 6-phosphate (Man-6-P) receptors (MPRs), the cation-dependent MPR (CD-MPR) and the insulin-like growth factor II/MPR (IGF-II/MPR), recognize a diverse population of Man-6-P-containing ligands. The IGF-II/MPR is a type I transmembrane glycoprotein with a large extracytoplasmic region composed of 15 repeating domains that display sequence identity to each other and to the single extracytoplasmic domain of the CD-MPR. A structure-based sequence alignment of the two distinct Man-6-P-binding sites of the IGF-II/MPR with the CD-MPR implicates several residues of IGF-II/MPR domains 3 and 9 as essential for Man-6-P binding. To test this hypothesis single amino acid substitutions were made in constructs encoding either the N- or the C-terminal Man-6-P-binding sites of the bovine IGF-II/MPR. The mutant IGF-II/MPRs secreted from COS-1 cells were analyzed by pentamannosyl phosphate-agarose affinity chromatography, identifying four residues (Gln-392, Ser-431, Glu-460, and Tyr-465) in domain 3 and four residues (Gln-1292, His-1329, Glu-1354, and Tyr-1360) in domain 9 as essential for Man-6-P recognition. Binding affinity studies using the lysosomal enzyme, beta-glucuronidase, confirmed these results. Together these analyses provide strong evidence that the two Man-6-P-binding sites of the IGF-II/MPR are structurally similar to each other and to the CD-MPR and utilize a similar carbohydrate recognition mechanism.

Highlights

Lysosomes, membranous organelles containing numerous acid hydrolases, play an essential role in the degradative metabolism of mammalian tissues and cells
Targeting of newly synthesized acid hydrolases to lysosomes is dependent upon specific recognition in the trans-Golgi network of mannose 6-phosphate (Man-6-P)1 residues found on the N-linked oligosaccharides of lysosomal enzymes by the two members of the P-type lectin family, the cation-dependent Man-6-P receptor (CD-MPR) and the insulin-like growth factor II/Man-6-P recep
Site-directed Mutagenesis of the N- and C-terminal Man-6P-binding Sites of the IGF-II/MPR—A structure-based sequence alignment of the extracytoplasmic region of the CDMPR with domains 3 and 9 of the IGF-II/MPR and molecular modeling identified several residues in domain 3 (Tyr-368, Gln-392, Ser-430, Ser-431, Arg-435, Thr-458, Glu-460, and Tyr465) and domain 9 (Tyr-1264, Gln-1292, His-1329, Arg-1334, Leu-1352, Glu-1354, and Tyr-1360) that are located at positions equivalent to cation-dependent MPR (CD-MPR) residues that serve as potential hydrogen bond donors/acceptors with Man-6-P (Fig. 2A) [34]

Summary

The abbreviations used are

Man-6-P, mannose 6-phosphate; MPR, mannose 6-phosphate receptor; IGF-II, insulin-like growth factor II; CD-MPR, cation-dependent mannose 6-phosphate receptor; IGF-II/ MPR, insulin-like growth factor II/mannose 6-phosphate receptor; Ni-NTA, nickel-nitrilotriacetic acid; TEMED, N,N,N,NЈ-tetramethylethylenediamine. Sitedirected mutagenesis was used to generate single amino acid substitutions in soluble, truncated forms of the IGFII/MPR in order to test the hypothesis that residues of IGF-II/MPR extracytoplasmic domains 3 and 9 predicted by a structure-based sequence alignment with the CD-MPR to be in the N- and C-terminal Man-6-P-binding pockets of the IGF-II/MPR, respectively, are essential for high affinity Man-6-P binding. To assess quantitatively the relative contribution of each of the residues essential to the Man-6-P binding ability of the IGF-II/MPR, binding affinity studies of the mutant constructs for the lysosomal enzyme, ␤-glucuronidase, were performed Together these results provide strong evidence that the two IGF-II/MPR Man-6-P-binding sites utilize a mechanism similar to that of the CD-MPR for high affinity Man-6-P binding and that the N- and C-terminal carbohydrate recognition domains of the IGF-II/MPR are structurally similar to each other and to the CD-MPR

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION