Biochemical and functional properties of the full-length cation-dependent mannose 6-phosphate receptor expressed in Pichia pastoris. Biochem Biophys Res Commun 2003 Sep 26;309(3):643-51
Date
09/10/2003Pubmed ID
12963039DOI
10.1016/j.bbrc.2003.08.048Scopus ID
2-s2.0-0041328866 (requires institutional sign-in at Scopus site) 7 CitationsAbstract
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.
Author List
Reddy ST, Kumar SN, Haas AL, Dahms NMAuthor
Nancy M. Dahms PhD Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Amino Acid SequenceAnimals
Asparagine
Binding, Competitive
Cattle
Gene Expression
Glycosylation
Molecular Sequence Data
Palmitic Acids
Pichia
Protein Processing, Post-Translational
Receptor, IGF Type 2
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Transformation, Genetic
