Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris.
A glycosylation-deficient, full-length cation-dependent mannose 6-phosphate receptor (CD-MPR) containing a yeast signal sequence was expressed in Pichia pastoris using the constitutive promoter of the PGAP gene. The membrane-bound receptor was solubilized using detergents and purified by pentamannosyl phosphate-agarose affinity chromatography. Equilibrium binding studies identified a binding affinity of 2 nM for the lysosomal enzyme, beta-glucuronidase. To probe the linkage specificity of the recombinant CD-MPR, inhibition binding studies were conducted using non-phosphorylated oligomannoses which demonstrated that Manalpha1,2Man exhibits a 4-fold higher inhibition than Manalpha1,3Man and Manalpha1,6Man. The receptor was capable of associating into oligomeric forms and enzymatic deglycosylation revealed the presence of high-mannose sugars at the single potential N-glycosylation site. Mass spectrometric analysis revealed that the receptor was palmitoylated at the two potential cysteines in its cytoplasmic domain. In conclusion, the full-length CD-MPR produced in P. pastoris is structurally and functionally suitable for crystallization studies.
Reddy
Sreelatha T
ST
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Kumar
Suresh N
SN
Haas
Arthur L
AL
Dahms
Nancy M
NM
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001667:Binding, Competitive; D002417:Cattle; D015870:Gene Expression; D006031:Glycosylation; D008969:Molecular Sequence Data; D010169:Palmitic Acids / Q000032:analysis; D010843:Pichia / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014170:Transformation, Genetic
D010169:Palmitic Acids; D017527:Receptor, IGF Type 2; D001216:Asparagine