Characterization of truncated and glycosylation-deficient forms of the cation-dependent mannose 6-phosphate receptor expressed in baculovirus- infected insect cells

Patricia G. Marron-Terada, Kathryn Elizabeth Bollinger, Nancy M. Dahms

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 crosslinking 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 K(d) of Stop155 and Asn81/Stop155 for the lysosomal enzyme,/β-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.

Original languageEnglish (US)
Pages (from-to)17223-17229
Number of pages7
JournalBiochemistry
Volume37
Issue number49
DOIs
StatePublished - Dec 8 1998

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Glycosylation
Baculoviridae
Insects
Oligosaccharides
Ligands
Affinity chromatography
Agarose Chromatography
Dimerization
Glucuronidase
Cytoplasmic and Nuclear Receptors
Affinity Chromatography
Recombinant Proteins
Crosslinking
Sepharose
Gel Chromatography
Sucrose
Gels
Phosphates
Acids
cation-dependent mannose-6-phosphate receptor

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of truncated and glycosylation-deficient forms of the cation-dependent mannose 6-phosphate receptor expressed in baculovirus- infected insect cells. / Marron-Terada, Patricia G.; Bollinger, Kathryn Elizabeth; Dahms, Nancy M.

In: Biochemistry, Vol. 37, No. 49, 08.12.1998, p. 17223-17229.

Research output: Contribution to journalArticle

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