Abstract
Macrophages express a cell surface receptor which mediates phagocytosis and pinocytosis of particles and solutes containing mannose (fucose and N-acetylglucosamine are also ligands for the receptor). An apparently identical protein has been isolated from human placenta. Proteolytic fragments of the placental receptor were sequenced so that oligonucleotide probes complementary to the receptor cDNA could be generated. These probes were used to isolate cDNA clones covering the entire coding portion of the mRNA for the receptor. Confirmation that these clones encode the mannose receptor was obtained by expression in rat fibroblasts. The expressed protein mediates uptake and degradation of mannose-conjugated serum albumin. The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH terminus. The extracellular portion of the receptor polypeptide consists of three types of domains. The first 139 amino acids constitute a cysteine-rich segment which does not resemble other known sequences. There follows a domain which closely resembles fibronectin type II repeats. The remainder of the extracellular portion of the receptor is composed of eight segments homologous with the C-type carbohydrate-recognition domains of the asialoglycoprotein receptor, mannose binding proteins, and other Ca2(+)-dependent animal lectins. This structure suggests that the receptor may contain multiple ligand-binding domains thus accounting for its tight binding to highly multivalent ligands.
Highlights
From the SDepartment of Biochemistry and Molecular Biophysics, Columbia University, $Department of Cell Biology and Physiology, Washington University School of Medicine, D
The deduced amino acid sequence of the receptor reveals that it is most likely to be a type I transmembrane protein (COOH terminus on the cytoplasmic side of the membrane) since the mature polypeptide is preceded by a signal sequence and a hydrophobic stop transfer sequence is located 45 amino acids from the COOH
Isolation of cDNA Clones for Mannose Receptor-With the quantities of protein available from human placenta, it was possible to determine the NH&erminal amino acid sequence as well as sequences of a number of peptides generated by trypsin, clostripain, and protease Arg-C digestion
Summary
Materials-Human placental mRNA and two human placental cDNA libraries were obtained from Clonetech, Inc. (Palo Alto, CA). Edman degradation on Applied Biosystems 470A and 477A Sequencers, and phenylthiohydantoin derivatives of amino acids were identified by reversed-phase high-performance liquid chromatography. In subsequent screenings using restriction fragment probes, fragments were labeled using [a-“‘P]dCTP, random primers, and the Klenow fragment of DNA polymerase, following Amersham. The cell medium (Dulbecco’s modified Eagle’s medium containing 10% newborn calf serum) was changed after 24 h, harvested after a further 48 h of growth, and filtered through a 0.45~Frn filter. Pseudovirus isolated in this way were used to infect Rat 6 fibroblasts [23] for 2 h in the presence of 8 fig/ml polybrene. Analysis of uptake and degradation of ‘251-labeled mannoseconjugated bovine serum albumin followed the protocol previously used for analysis of the chicken hepatic lectin [25], except that specificity was measured using an excess of unlabeled ligand
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