Publication Date:
2015-12-27
Description:
The acquisition of mannose 6-phosphate (Man6 P ) 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 Man6 P monosaccharide that, through an array-screening approach against a number of phosphorylated N -glycans, is shown to bind mono- and diphosphorylated Man 6 and Man 7 glycans that contain terminal αMan6 P (1 -〉 2)αMan(1 -〉 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man 8 or mono- or diphosphorylated Man 9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6 P 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 p K a of Man6 P (p K a = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6 P 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 Man6 P recognition.
Print ISSN:
0959-6658
Electronic ISSN:
1460-2423
Topics:
Biology
,
Medicine
Permalink
