Identification of residues essential for carbohydrate recognition and cation dependence of the 46-kDa mannose 6-phosphate receptor.
The 46 kDa cation-dependent mannose 6-phosphate receptor (CD-MPR) plays an essential role in the biogenesis of lysosomes by diverting newly synthesized mannose 6-phosphate (Man-6-P)-containing lysosomal enzymes from the secretory pathway to acidified endosomes. Previous crystallographic studies of the CD-MPR have identified 11 amino acids within its carbohydrate binding pocket. These residues were evaluated quantitatively by assaying the binding affinity of mutant receptors containing a single amino acid substitution toward a lysosomal enzyme. The results show that substitution of Gln-66, Arg-111, Glu-133, or Tyr-143 results in a >800-fold decrease in affinity, demonstrating these four amino acids are essential for carbohydrate recognition by the CD-MPR. Solution binding and surface plasmon resonance analyses demonstrated that the presence of Mn2+ enhanced the affinity of the CD-MPR for a lysosomal enzyme by 2- to 4-fold and increased the stoichiometry of the interaction between a heterogeneous population of a lysosomal enzyme and the receptor by approximately 3-fold. In contrast, substitution of Asp-103 results in a protein that no longer exhibits enhanced binding affinities or altered stoichiometry in the presence of cations, and electron spin resonance demonstrated that the D103S mutant exhibits a 6-fold lower affinity for Mn2+ than the wild-type receptor (Kd = 3.7 6 1.4 mM versus 0.6 6 0.1 mM). Chemical cross-linking revealed that Mn2+ influences the stoichiometry of interaction between the CD-MPR and lysosomal enzymes by increasing the oligomeric state of the receptor from dimer to higher order oligomers. Taken together, these studies provide the molecular basis for high affinity carbohydrate recognition by the CD-MPR. Furthermore, Asp-103 has been identified as the key residue which mediates the effects of divalent cations on the binding properties of the CD-MPR.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Guangjie
G
Sun
Hongtao
H
Zhao
B
B
Kalyanaraman
Nancy M
NM
Dahms
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D050260:Carbohydrate Metabolism; D002412:Cations / Q000737:chemistry; D002417:Cattle; D004285:Dogs; D004578:Electron Spin Resonance Spectroscopy; D005966:Glucuronidase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000502:physiology; D008247:Lysosomes / Q000201:enzymology / Q000378:metabolism; D008345:Manganese / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000502:physiology; D020349:Surface Plasmon Resonance; D013997:Time Factors
D002412:Cations; D037102:Lectins; D011956:Receptors, Cell Surface; D001224:Aspartic Acid; D008345:Manganese; D006639:Histidine; D001216:Asparagine; D005966:Glucuronidase