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A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2.

Jégouzo SA, Feinberg H, Dungarwalla T, Drickamer K, Weis WI, Taylor ME - J. Biol. Chem. (2015)

Bottom Line: X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca(2+) in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site.As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins.Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.

View Article: PubMed Central - PubMed

Affiliation: the Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.

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Purification of the CRD from BDCA-2 by affinity chromatography.A, the CRD was renatured from inclusion bodies by dialysis against Ca2+-containing buffer followed by dialysis against water and lyophilization to obtain a concentrated sample that was applied to a 10-ml column of mannose-Sepharose. After application of the sample, the column was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2. Fractions of 2 ml were collected. B, following renaturation by dialysis, the CRD was applied directly to a 5-ml column of desialylated egg yolk glycopeptide immobilized on agarose. The column was rinsed with 25 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2, and protein was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 2.5 mm EDTA in 1-ml fractions. In both cases, aliquots (15 μl) from fractions were analyzed on SDS-polyacrylamide gels that were stained with Coomassie Blue. The expected molecular weight of the BDCA-2 CRD is 17,100.
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Figure 2: Purification of the CRD from BDCA-2 by affinity chromatography.A, the CRD was renatured from inclusion bodies by dialysis against Ca2+-containing buffer followed by dialysis against water and lyophilization to obtain a concentrated sample that was applied to a 10-ml column of mannose-Sepharose. After application of the sample, the column was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2. Fractions of 2 ml were collected. B, following renaturation by dialysis, the CRD was applied directly to a 5-ml column of desialylated egg yolk glycopeptide immobilized on agarose. The column was rinsed with 25 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2, and protein was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 2.5 mm EDTA in 1-ml fractions. In both cases, aliquots (15 μl) from fractions were analyzed on SDS-polyacrylamide gels that were stained with Coomassie Blue. The expected molecular weight of the BDCA-2 CRD is 17,100.

Mentions: Following expression of the CRD from BDCA-2 in E. coli as inclusion bodies, several protocols for renaturation were investigated, and attempts were made to purify the CRD on high density mannose-Sepharose resin, by analogy to protocols used for mincle and other C-type lectins. When renatured protein was applied to an extended affinity column in a small sample volume, retardation of a band corresponding to the CRD from BDCA-2 was observed (Fig. 2A). Although the protein did not stick tightly to the column and washed through in the presence of Ca2+, the results provide evidence that BDCA-2 has at least weak mannose binding activity.


A Novel Mechanism for Binding of Galactose-terminated Glycans by the C-type Carbohydrate Recognition Domain in Blood Dendritic Cell Antigen 2.

Jégouzo SA, Feinberg H, Dungarwalla T, Drickamer K, Weis WI, Taylor ME - J. Biol. Chem. (2015)

Purification of the CRD from BDCA-2 by affinity chromatography.A, the CRD was renatured from inclusion bodies by dialysis against Ca2+-containing buffer followed by dialysis against water and lyophilization to obtain a concentrated sample that was applied to a 10-ml column of mannose-Sepharose. After application of the sample, the column was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2. Fractions of 2 ml were collected. B, following renaturation by dialysis, the CRD was applied directly to a 5-ml column of desialylated egg yolk glycopeptide immobilized on agarose. The column was rinsed with 25 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2, and protein was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 2.5 mm EDTA in 1-ml fractions. In both cases, aliquots (15 μl) from fractions were analyzed on SDS-polyacrylamide gels that were stained with Coomassie Blue. The expected molecular weight of the BDCA-2 CRD is 17,100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4505424&req=5

Figure 2: Purification of the CRD from BDCA-2 by affinity chromatography.A, the CRD was renatured from inclusion bodies by dialysis against Ca2+-containing buffer followed by dialysis against water and lyophilization to obtain a concentrated sample that was applied to a 10-ml column of mannose-Sepharose. After application of the sample, the column was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2. Fractions of 2 ml were collected. B, following renaturation by dialysis, the CRD was applied directly to a 5-ml column of desialylated egg yolk glycopeptide immobilized on agarose. The column was rinsed with 25 ml of 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 25 mm CaCl2, and protein was eluted with 150 mm NaCl, 25 mm Tris-Cl, pH 7.8, 2.5 mm EDTA in 1-ml fractions. In both cases, aliquots (15 μl) from fractions were analyzed on SDS-polyacrylamide gels that were stained with Coomassie Blue. The expected molecular weight of the BDCA-2 CRD is 17,100.
Mentions: Following expression of the CRD from BDCA-2 in E. coli as inclusion bodies, several protocols for renaturation were investigated, and attempts were made to purify the CRD on high density mannose-Sepharose resin, by analogy to protocols used for mincle and other C-type lectins. When renatured protein was applied to an extended affinity column in a small sample volume, retardation of a band corresponding to the CRD from BDCA-2 was observed (Fig. 2A). Although the protein did not stick tightly to the column and washed through in the presence of Ca2+, the results provide evidence that BDCA-2 has at least weak mannose binding activity.

Bottom Line: X-ray crystallography and mutagenesis studies show that mannose is ligated to the conserved Ca(2+) in the primary binding site that is characteristic of C-type carbohydrate recognition domains, and the GlcNAc and galactose residues make additional interactions in a wide, shallow groove adjacent to the primary binding site.As predicted from these studies, BDCA-2 binds to IgG, which bears galactose-terminated glycans that are not commonly found attached to other serum glycoproteins.Thus, BDCA-2 has the potential to serve as a previously unrecognized immunoglobulin Fc receptor.

View Article: PubMed Central - PubMed

Affiliation: the Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.

Show MeSH