STRA6, a Cell-Surface Receptor for Retinol-Binding Protein: The Plot Thickens

Members of the lipocalin superfamily share a common structural fold, but differ from each other with respect to the molecules with which they interact. They all contain eight β-strands (A—H) that fold to form a well-defined β-barrel, which harbours a binding pocket for hydrophobic ligands. These strands are connected by loops that vary in size and structure and make up the closed and open ends of the pocket. In addition to binding ligands, some members of the family interact with other macromolecules, the specificity of which is thought to be associated with the variable loop regions. Here, we have investigated whether the macromolecular-recognition properties can be transferred from one member of the family to another. For this, we chose the prototypical lipocalin, the plasma retinol-binding protein (RBP) and its close structural homologue the epididymal retinoic acid-binding protein (ERABP). RBP exhibits three molecular-recognition properties: it binds to retinol, to transthyretin (TTR) and to a cell-surface receptor. ERABP binds retinoic acid, but whether it interacts with other macromolecules is not known. Here, we show that ERABP does not bind to TTR and the RBP receptor, but when the loops of RBP near the open end of the pocket (L-1, L-2 and L-3, connecting β-strands A—B, C—D and E—F, respectively) were substituted into the corresponding regions of ERABP, the resulting chimaera acquired the ability to bind TTR and the receptor. L-2 and L-3 were found to be the major determinants of the receptor- and TTR-binding specificities respectively. Thus we demonstrate that lipocalins serve as excellent scaffolds for engineering novel biological functions.

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Vitamin A Transport and the Transmembrane Pore in the Cell-Surface Receptor for Plasma Retinol Binding Protein

PLoS ONE ◽

10.1371/journal.pone.0073838 ◽

2013 ◽

Vol 8 (11) ◽

pp. e73838 ◽

Cited By ~ 14

Author(s):

Ming Zhong ◽

Riki Kawaguchi ◽

Mariam Ter-Stepanian ◽

Miki Kassai ◽

Hui Sun

Keyword(s):

Vitamin A ◽

Cell Surface ◽

Binding Protein ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Surface Receptor ◽

Plasma Retinol ◽

Vitamin A Transport

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A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish

Journal of Cell Science ◽

10.1242/jcs.112.6.917 ◽

1999 ◽

Vol 112 (6) ◽

pp. 917-925

Author(s):

M.W. Johansson ◽

T. Holmblad ◽

P.O. Thornqvist ◽

M. Cammarata ◽

N. Parrinello ◽

...

Keyword(s):

Superoxide Dismutase ◽

Cell Surface ◽

Signal Sequence ◽

Binding Protein ◽

Cell Surface Receptor ◽

Cell Adhesive ◽

Surface Receptor ◽

A Cell ◽

Polymerase Chain ◽

Membrane Spanning

Peroxinectin, a cell-adhesive peroxidase (homologous to human myeloperoxidase), from the crayfish Pacifastacus leniusculus, was shown by immuno-fluorescence to bind to the surface of crayfish blood cells (haemocytes). In order to identify a cell surface receptor for peroxinectin, labelled peroxinectin was incubated with a blot of haemocyte membrane proteins. It was found to specifically bind two bands of 230 and 90 kDa; this binding was decreased in the presence of unlabelled peroxinectin. Purified 230/90 kDa complex also bound peroxinectin in the same assay. In addition, the 230 kDa band binds the crayfish beta-1,3-glucan-binding protein. The 230 kDa band could be reduced to 90 kDa, thus showing that the 230 kDa is a multimer of 90 kDa units. The peroxinectin-binding protein was cloned from a haemocyte cDNA library, using immuno-screening or polymerase chain reaction based on partial amino acid sequence of the purified protein. It has a signal sequence, a domain homologous to CuZn-containing superoxide dismutases, and a basic, proline-rich, C-terminal tail, but no membrane-spanning segment. In accordance, the 90 and 230 kDa bands had superoxide dismutase activity. Immuno-fluorescence of non-permeabilized haemocytes with affinity-purified antibodies confirmed that the crayfish CuZn-superoxide dismutase is localized at the cell surface; it could be released from the membrane with high salt. It was thus concluded that the peroxinectin-binding protein is an extracellular SOD (EC-SOD) and a peripheral membrane protein, presumably kept at the cell surface via ionic interaction with its C-terminal region. This interaction with a peroxidase seems to be a novel function for an SOD. The binding of the cell surface SOD to the cell-adhesive/opsonic peroxinectin may mediate, or regulate, cell adhesion and phagocytosis; it may also be important for efficient localized production of microbicidal substances.

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Structure-function studies on human retinol-binding protein using site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj3000437 ◽

1994 ◽

Vol 300 (2) ◽

pp. 437-442 ◽

Cited By ~ 24

Author(s):

A Sivaprasadarao ◽

J B Findlay

Keyword(s):

Cell Surface ◽

Specific Binding ◽

Binding Protein ◽

Specific Interaction ◽

Binding Activity ◽

Site Directed Mutagenesis ◽

Retinol Binding Protein ◽

Cell Surface Receptor ◽

Directed Mutagenesis ◽

Surface Receptor

Retinol-binding protein (RBP) transports vitamin A in the plasma. It consists of eight anti-parallel beta-strands (A to H) that fold to form an orthogonal barrel. The loops connecting the strands A and B, C and D, and E and F form the entrance to the binding site in the barrel. The retinol molecule is found deep inside this barrel. Apart from its specific interaction with retinol, RBP is involved in two other molecular-recognition properties, that is it binds to transthyretin (TTR), another serum protein, and to a cell-surface receptor. Using site-directed mutagenesis, specific changes were made to the loop regions of human RBP and the resultant mutant proteins were tested for their ability to bind to retinol, to TTR and to the RBP receptor. While all the variants retained their ability to bind retinol, that in which residues 92 to 98 of the loop E-F were deleted completely lost its ability to interact with TTR, but retained some binding activity for the receptor. In contrast, the double mutant in which leucine residues at positions 63 and 64 of the loop C-D were changed to arginine and serine respectively partially retained its TTR-binding ability, but completely lost its affinity for the RBP receptor. Mutation of Leu-35 of loop A-B to valine revealed no apparent effect on any of the binding activities of RBP. However, substitution of leucine for proline at position 35 markedly reduced the affinity of the protein for TTR, but showed no apparent change in its receptor-binding activity. These results demonstrate that RBP interacts with both TTR and the receptor via loops C-D and E-F. The binding sites, however, are overlapping rather than identical. RBP also appears to make an additional contact with TTR via its loop A-B. A further implication of these results is that RBP, when bound to TTR, cannot bind simultaneously to the receptor. This observation is consistent with our previously proposed mechanism for delivery of retinol to target tissues [Sivaprasadarao and Findlay (1988) Biochem. J. 255, 571-579], according to which retinol delivery involves specific binding of RBP to the cell-surface receptor, an interaction that triggers release of retinol from RBP to the bound cell rather than internalization of retinol-RBP complex.

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