Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor

Abstract

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity (ϳ1 nM) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was ϳ300-fold lower (K i ‫؍‬ 5.3 mM) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme ␤-glucuronidase was also much lower (K d ‫؍‬ 54 M) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

Ligands for the β-Glucan Receptor, Dectin-1, Assigned Using “Designer” Microarrays of Oligosaccharide Probes (Neoglycolipids) Generated from Glucan Polysaccharides

“…not recognized by Dectin-1. Among them are oligosaccharide probes recognized by other mammalian carbohydrate-binding proteins (21,31,42). Here, the ability to generate tailor-made oligosaccharide probes from targeted sources, fungal and bacterial polysaccharides, in conjunction with MS and methylation analysis has been crucial for assigning oligosaccharide ligands for Dectin-1.…”

Identification of the Insulin-Like Growth Factor II Receptor as a Novel Receptor for Binding and Invasion by Listeria monocytogenes

“…The peptide sequence identified in our studies is located in repeat 3 of the extracellular domain that is one of the three M6P binding sites of the IGFIIR (25,68). Recently, the amino acid sequence in repeat 3 was determined (25,38,65); the identified (M6Pbs) sequence is located between two conserved cysteine residues (C334 and C375) and upstream of VOL.…”

“…Recently, the amino acid sequence in repeat 3 was determined (25,38,65); the identified (M6Pbs) sequence is located between two conserved cysteine residues (C334 and C375) and upstream of VOL. 74,2006 IGFIIR IS A NOVEL RECEPTOR FOR LISTERIA 573 a conserved residue (Y368) which is essential for carbohydrate recognition (38,65,68,84). Adherence of Listeria to mammalian cells is known to occur through a wide range of different listerial virulence factors and cell surface components such as fibronectin binding proteins (31,41), carbohydrates (15,20,22,67), protein 60 (9,72), the autolysin ami (58), lipoproteins (69), and teichoic and lipoteichoic acids (1,34,66,67).…”

Production of Recombinant β-Hexosaminidase A, a Potential Enzyme for Replacement Therapy for Tay-Sachs and Sandhoff Diseases, in the Methylotrophic Yeast Ogataea minuta

“…Dom9His was expressed and purified according to the method described by Reddy et al (36). All chromatograms were performed at 4°C under the recommended conditions for the respective columns.…”