Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishesN-glycan phosphorylation in a branch-specific manner

Abstract

The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition.

Targeted Analysis of Lysosomal Directed Proteins and Their Sites of Mannose-6-phosphate Modification

“…When compared with scans that contained only 204 ion and no 243 ion, around 200 scans, we calculated that over 90% of all glycopeptide scans are in fact originating from M6P-harboring glycopeptides. Our findings support in vitro studies, which revealed that the Man7 PP glycoform exhibits the highest binding affinity toward mannose-6-phosphate receptors (30,31). We also further validated our results by performing a pathway enrichment analysis (32), which showed, as expected, significant enrichment for lysosomal proteins (Fig.…”

Influenza binds phosphorylated glycans from human lung

“…We confirmed that alkaline phosphatase was efficient, using a synthetic glycan array containing a few phosphorylated glycans, in which alkaline phosphatase caused loss of binding of the single-chain variable domain antibody fragment (scFv) M6P-1, specific for selected mannose-6-phosphate (Man6P)–containing glycans (fig. S6) ( 19 ). Notably, the dephosphorylation of the HL-SGM by alkaline phosphatase treatment following NA treatment resulted in a total loss of virus binding (Fig.…”

Chimeric oligosaccharide conjugate induces opsonic antibodies against Streptococcus pneumoniae serotypes 19A and 19F

“…The absence of the phosphate diester in the shorter antigens might impair the generation of antibodies that can recognize native CPS since Prevnar13®, that contains the native repeating units (RU) in varying length, is able to induce a high immune response. Antibodies raised against ST19A and ST19F did not cross react showing the importance of L-rhamnose linkage 48,49 in generating specic antibodies against native CPS of different bacteria (Fig. 3).…”

“…Increased binding to the chimeric oligosaccharide may be a result of an ionic interaction with the phosphate diester connecting the two repeating units. Ionic protein-glycan interactions are known for cell recognition 48,49 and antibody-glycan interactions. 15 Rabbit antibodies raised in response to glycoconjugate vaccination were tested against native CPS of ST19A (CPS19A) and ST19F (CPS19F) to determine the cross-reactivity of antibodies produced against synthetic or native antigens.…”