Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.
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 ( approximately 1 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 approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) 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.
Reddy
Sreelatha T
ST
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Chai
Wengang
W
Childs
Robert A
RA
Page
Jimmy D
JD
Feizi
Ten
T
Dahms
Nancy M
NM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D004305:Dose-Response Relationship, Drug; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008247:Lysosomes / Q000378:metabolism; D008360:Mannosephosphates / Q000737:chemistry*; D015233:Models, Statistical; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D009844:Oligosaccharides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017527:Receptor, IGF Type 2 / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D002241:Carbohydrates; D002412:Cations; D003432:Cross-Linking Reagents; D004220:Disulfides; D008024:Ligands; D008360:Mannosephosphates; D009844:Oligosaccharides; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D006026:Glycoside Hydrolases