Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 277, Issue 49, Pages 47205-47212Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M208534200
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- NIDDK NIH HHS [DK42667] Funding Source: Medline
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The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, beta-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K-d=0.3+/-0.1 nM) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K(d)similar to500 nM) toward beta-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.
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