Expression and Characterization of Functional Bovine Cation-Dependent Mannose 6-Phosphate Receptors in Baculovirus-Infected Insect Cells

Dahms, Nancy M.; Brzycki-Wessell, Mary A.

doi:10.1006/abbi.1995.1193

Cited by 10 publications

(3 citation statements)

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“…teins (32)(33) in spite of the fact that their glycosylation CE analysis (data not shown) demonstrated that pattern differs from the native glycoprotein. nC1INH and rC1INH samples contained similar Examination of the C1INH samples was undertaken amounts of O-glycans.…”

Section: Analysis Of C1inh O-glycansmentioning

confidence: 57%

Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization

Wolff

Zhang

Roberg

et al. 2001

Protein Expression and Purification

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Section: Analysis Of C1inh O-glycansmentioning

confidence: 57%

Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization

Wolff

Zhang

Roberg

et al. 2001

Protein Expression and Purification

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“…Expression and Purification of Truncated IGF-II/MPRs . Previous work in our laboratory determined that S. frugiperda (Sf9) and T. ni 5B1-4 (High Five) insect cells do not contain endogenous MPRs, and that insect cells infected with recombinant baculovirus encoding the CD-MPR produce functional receptors ( , ). To obtain sufficient quantities of protein to carry out biochemical analyses of the CRDs of the IGF-II/MPR, recombinant baculovirus encoding the various Dom 1−3, Dom 7−9, or Dom 7−11 constructs was used to infect High Five insect cells, and the cells and medium were harvested 4−5 days postinfection.…”

Section: Resultsmentioning

confidence: 99%

Recognition of Dictyostelium discoideum Lysosomal Enzymes Is Conferred by the Amino-Terminal Carbohydrate Binding Site of the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

et al. 2000

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The insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/MPR) is a type I glycoprotein that mediates both the intracellular sorting of lysosomal enzymes bearing mannose 6-phosphate (Man-6-P) residues to the lysosome and the bioavailability of IGF-II. The extracytoplasmic region of the IGF-II/MPR contains 15 repeating domains; the two carbohydrate recognition domains (CRDs) have been localized to domains 1-3 and 7-9, and the high-affinity IGF-II binding site maps to domain 11. To characterize the carbohydrate binding properties of the IGF-II/MPR, regions of the receptor encompassing the individual CRDs were produced in a baculovirus expression system. Characterization of the recombinant proteins revealed that the pH optimum for carbohydrate binding is significantly more acidic for the carboxyl-terminal CRD than for the amino-terminal CRD (i.e., pH 6.4-6.5 vs 6.9). Equilibrium binding studies demonstrated that the two CRDs exhibit a similar affinity for Man-6-P. Furthermore, substitution of the conserved arginine residue in domain 3 (R435) or in domain 9 (R1334) with alanine resulted in a similar >1000-fold decrease in the affinity for the lysosomal enzyme, beta-glucuronidase. In contrast, the two CRDs differ dramatically in their ability to recognize the distinctive modifications (i.e., mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes: the amino-terminal CRD binds mannose 6-sulfate and Man-6-P methyl ester with a 14-55-fold higher affinity than the carboxyl-terminal CRD. Taken together, these results demonstrate that the IGF-II/MPR contains two functionally distinct CRDs.

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“…After attachment, cells are washed with PBS and fixed by incubation on ice for 20 min in the presence of 10% formalin in PBS. Cells were rinsed with PBS plus 0.1% BSA before being incubated overnight at 4 °C in the dark in a solution of PBS plus 0.1% BSA and a 1:100 dilution of anti-CD-MPR (NMD) [4]. Cells were then rinsed with PBS plus 0.1% BSA two times prior to incubation with a 1:500 chicken anti-rabbit Alexa 864 antibody.…”

Section: Figmentioning

confidence: 99%

Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System

Olson

Dahms

2017

Methods in Molecular Biology

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The cation-dependent mannose 6-phosphate receptor (CD-MPR) is a single-pass type I membrane protein. This protein functions to transport lysosomal enzymes displaying phosphomannosyl residues from the Golgi complex and the cell surface to the lysosome. This glycosylated protein contains three disulfide bridges in its 159-residue extracytoplasmic domain. One of the problems with studying eukaryotic membrane proteins is isolating sufficient quantities. Structural studies typically require several hundred milligrams of highly purified protein. Here we present a method to isolate milligram quantities of CD-MPR/Asn81 suitable for structural studies.

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Expression and Characterization of Functional Bovine Cation-Dependent Mannose 6-Phosphate Receptors in Baculovirus-Infected Insect Cells

Cited by 10 publications

References 0 publications

Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization

Expression of C1 Esterase Inhibitor by the Baculovirus Expression Vector System: Preparation, Purification, and Characterization

Recognition of Dictyostelium discoideum Lysosomal Enzymes Is Conferred by the Amino-Terminal Carbohydrate Binding Site of the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor

Cloning, Expression, and Purification of the Glycosylated Transmembrane Protein, Cation-Dependent Mannose 6-Phosphate Receptor, from Sf9 Cells Using the Baculovirus System

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