Asymmetry adjacent to the collagen-like domain in rat liver mannose-binding protein

Rat liver mannose-binding protein (MBP-C) is the smallest known member of the collectin family of animal lectins, many of which are involved in defence against microbial pathogens. It consists of an N-terminal collagen-like domain linked to C-terminal carbohydrate-recognition domains. MBP-C, overproduced in Chinese-hamster ovary cells, is post-translationally modified and processed in a manner similar to the native lectin. Analytical ultracentrifugation experiments indicate that MBP-C is trimeric, with a weight-averaged molecular mass of approx. 77 kDa. The rate of sedimentation of MBP-C and its mobility on gel filtration suggest a highly elongated molecule. Anomalous behaviour on gel filtration due to this extended conformation may explain previous suggestions that MBP-C forms a higher oligomer. The polypeptide chains of the MBP-C trimer are linked by disulphide bonds between two cysteine residues at the N-terminal junction of the collagen-like domain. Analysis of an N-terminal tryptic fragment reveals that the disulphide bonding in MBP-C is heterogeneous and asymmetrical. These results indicate that assembly of MBP-C oligomers probably proceeds in a C- to N-terminal direction: trimerization at the C-terminus is followed by assembly of the collagenous domain and finally formation of N-terminal disulphide bonds. The relatively simple organization of MBP-C provides a template for understanding larger, more complex collectins.

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Structural Analysis of Monosaccharide Recognition by Rat Liver Mannose-binding Protein

Journal of Biological Chemistry ◽

10.1074/jbc.271.2.663 ◽

1996 ◽

Vol 271 (2) ◽

pp. 663-674 ◽

Cited By ~ 123

Author(s):

Kenneth K.-S. Ng ◽

Kurt Drickamer ◽

William I. Weis

Keyword(s):

Structural Analysis ◽

Rat Liver ◽

Binding Protein ◽

Mannose Binding Protein ◽

Mannose Binding

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Orientation of sugars bound to the principal C-type carbohydrate-recognition domain of the macrophage mannose receptor

Biochemical Journal ◽

10.1042/bj3330601 ◽

1998 ◽

Vol 333 (3) ◽

pp. 601-608 ◽

Cited By ~ 23

Author(s):

Paul G. HITCHEN ◽

Nicholas P. MULLIN ◽

Maureen E. TAYLOR

Keyword(s):

Binding Site ◽

Rat Liver ◽

Binding Protein ◽

Mannose Receptor ◽

Tyrosine Residue ◽

Carbohydrate Recognition ◽

Sugar Binding ◽

Macrophage Mannose Receptor ◽

Mannose Binding Protein ◽

Mannose Binding

The extracellular region of the macrophage mannose receptor, a protein involved in the innate immune response, contains eight C-type carbohydrate-recognition domains (CRDs). The fourth of these domains, CRD-4, is central to ligand binding by the receptor, and binds mannose, fucose and N-acetylglucosamine by direct ligation to Ca2+. Site-directed mutagenesis combined with NMR and molecular modelling have been used to determine the orientation of monosaccharides bound to CRD-4. Two resonances in the 1H NMR spectrum of CRD-4 that are perturbed on sugar binding are identified as a methyl proton from a leucine side chain in the core of the domain and the H-2 proton of a histidine close to the predicted sugar-binding site. The effects of mutagenesis of this histidine residue, a nearby isoleucine residue and a tyrosine residue previously shown to stack against sugars bound to CRD-4 show the absolute orientation of sugars in the binding site. N-Acetylglucosamine binds to CRD-4 of the mannose receptor in the orientation seen in crystal structures of the CRD of rat liver mannose-binding protein. Mannose binds to CRD-4 in the orientation seen in the CRD of rat serum mannose-binding protein and is rotated by 180 ° relative to GlcNAc bound to CRD-4. Interaction of the O-methyl group and C-1 of α-methyl Fuc with the tyrosine residue accounts for the strong preference of CRD-4 for this anomer of fucose. Both anomers of fucose bind to CRD-4 in the orientation seen in rat liver mannose-binding protein.

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Isolation of mannose-binding proteins from human and rat liver

Biochemical Journal ◽

10.1042/bj2100167 ◽

1983 ◽

Vol 210 (1) ◽

pp. 167-174 ◽

Cited By ~ 55

Author(s):

J Wild ◽

D Robinson ◽

B Winchester

Keyword(s):

Affinity Chromatography ◽

Rat Liver ◽

Human Liver ◽

Binding Proteins ◽

Binding Assay ◽

Binding Protein ◽

Mannose Binding Protein ◽

Mannose Binding ◽

A Subunit ◽

Ribonuclease B

A binding assay for the detection of mannose-binding proteins was developed, which uses a ligand of mammalian origin, 125I-labelled bovine pancreatic ribonuclease B. The binding assay was validated by using the recognized mannose-binding protein, concanavalin A. Microgram quantities of concanavalin A or mannose-binding proteins could be assayed. A mannose-binding protein was isolated from rat liver by affinity chromatography on mannose-Sepharose 6B. It has a Mr of approx. 900000 under non-dissociating conditions and contains a subunit of approx. 34000 Mr. When ribonuclease B-Sepharose was used as a ligand for affinity chromatography, the predominant mannose-binding material isolated from rat liver had a native Mr of 205000-225000 and consisted largely of a subunit of Mr 70000, which yielded subunits of Mr 28500 and 34000 on reduction. It is suggested that different mannose-binding proteins are isolated by the two affinity-chromatography ligands. A mannose-binding protein was also purified from human liver by affinity chromatography on mannose-Sepharose 6B. It has a native Mr of over 1000000 and consists of subunits with Mr 28000 and 30500. Its isolation suggests that mannose-mediated endocytosis or intracellular transport of glycoproteins occurs in human liver.

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