Characterization of Truncated and Glycosylation-Deficient Forms of the Cation-Dependent Mannose 6-Phosphate Receptor Expressed in Baculovirus-Infected Insect Cells
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- 作者：Patricia G. Marron-Terada ; Kathryn E. Bollinger ; and Nancy M. Dahms
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：December 8, 1998
- 年：1998
- 卷：37
- 期：49
- 页码：17223 - 17229
- 全文大小：113K
- 年卷期：v.37,no.49(December 8, 1998)
- ISSN：1520-4995
文摘
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 theCD-MPR, Asn81/Stop155, which has been modified to contain only one N-linked glycosylation site atposition 81 instead of five, were purified from baculovirus-infected High Five insect cells. The glycosylatedrecombinant proteins were functional in ligand binding and acid-dependent dissociation as assessed bypentamannosyl phosphate-agarose affinity chromatography. Gel filtration, sucrose gradients, and cross-linking experiments revealed that both Stop155 and Asn81/Stop155 are dimeric, demonstrating that thetransmembrane and cytoplasmic region of the receptor as well as N-linked oligosaccharides at positions31, 57, and 87 are not required for dimerization. The Kd of Stop155 and Asn81/Stop155 for the lysosomalenzyme, 
-glucuronidase, was 0.2 and 0.3 nM, respectively. These values are very similar to those reportedfor the full-length CD-MPR, demonstrating that the extracellular region of the CD-MPR is sufficient forhigh-affinity binding and that oligosaccharides at positions 31, 57, and 87 do not influence ligand binding.
-glucuronidase, was 0.2 and 0.3 nM, respectively. These values are very similar to those reportedfor the full-length CD-MPR, demonstrating that the extracellular region of the CD-MPR is sufficient forhigh-affinity binding and that oligosaccharides at positions 31, 57, and 87 do not influence ligand binding.