Characterization of truncated and glycosylation-deficient forms of the cation-dependent mannose 6-phosphate receptor expressed in baculovirus-infected insect cells. Biochemistry 1998 Dec 08;37(49):17223-9
Date
12/23/1998Pubmed ID
9860836DOI
10.1021/bi981883yScopus ID
2-s2.0-0032497956 (requires institutional sign-in at Scopus site) 20 CitationsAbstract
A soluble truncated form of the cation-dependent mannose 6-phosphate receptor (CD-MPR) encoding only the extracytoplasmic region, Stop155, and a truncated glycosylation-deficient form of the CD-MPR, Asn81/Stop155, which has been modified to contain only one N-linked glycosylation site at position 81 instead of five, were purified from baculovirus-infected High Five insect cells. The glycosylated recombinant proteins were functional in ligand binding and acid-dependent dissociation as assessed by pentamannosyl phosphate-agarose affinity chromatography. Gel filtration, sucrose gradients, and cross-linking experiments revealed that both Stop155 and Asn81/Stop155 are dimeric, demonstrating that the transmembrane and cytoplasmic region of the receptor as well as N-linked oligosaccharides at positions 31, 57, and 87 are not required for dimerization. The Kd of Stop155 and Asn81/Stop155 for the lysosomal enzyme, beta-glucuronidase, was 0.2 and 0.3 nM, respectively. These values are very similar to those reported for the full-length CD-MPR, demonstrating that the extracellular region of the CD-MPR is sufficient for high-affinity binding and that oligosaccharides at positions 31, 57, and 87 do not influence ligand binding.
Author List
Marron-Terada PG, Bollinger KE, 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
AcidsAnimals
Asparagine
Binding Sites
Cations
Cattle
Cell Line
Chromatography, Affinity
Dimerization
Genetic Vectors
Glycosylation
Hydrogen-Ion Concentration
Mannosephosphates
Oligosaccharides
Peptide Fragments
Protein Isoforms
Receptor, IGF Type 2
Recombinant Proteins
Sepharose
Spodoptera
