Glycobiology Advance Access originally published online on March 6, 2003
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Glycobiology, 2003, Vol. 13, No. 5 12G-13G
© 2003 Oxford University Press
GLYCO-FORUM SECTION |
Letter to the Glyco-Forum
HARE raising
Department of Biochemistry and Molecular Biology and the Oklahoma Center for Medical Glycobiology, the University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA
Received on February 12, 2003; accepted on February 12, 2003
Smedsrod et al. (2003) have raised an issue regarding our study (Zhou et al., 2003
) on the human 190-kDa HA receptor for endocytosis (HARE), which appears in this issue of Glycobiology. We did not intend to slight their studies to characterize this HA receptor, which they term stabilin-2. However, we have a fundamentally different view about the type of the evidence needed to support a conclusion about the function of a newly discovered protein. We did not cite some conclusions from their previous studies because stating a conclusion in an article does not mean it is valid or fully supported by the data presented, as exemplified by the McCourt et al. (1994) study.
As with the early stages of many fields, the identification of the HA/chondroitin sulfate (CS) clearance receptor has had its share of misdirection and multiple discoveries of different protein activities and the same gene. Over the last few years, multiple proteins (some putative)—ultimately related to this endocytic HA receptor—have been described, for example, FEL-2, HA-scavenger receptor, stabilin-2, and HARE. FEL-2 was identified as a genomic sequence for a putative protein of unknown function that contained a Link domain (Jackson et al., 2002). The HA-scavenger receptor (McCourt et al., 1999) was purified from whole liver by a multistep procedure (using no assays to identify and track the protein being purified). Stabilin-2 was identified based on a proteomic approach using mass spectrometry analysis of peptides derived from a soluble form of the HA-scavenger receptor purified from whole liver (Politz et al., 2002). HARE was identified in liver sinusoidal endothelial cell membranes by purification using monoclonal antibodies and a functional assay to monitor HA-binding activity (Zhou et al., 1999).
Although these proteins are not all the same, the recent likely consensus is that they are derived from the same gene. No one yet knows how many discrete proteins may be derived from the large stabilin-2 gene. In fact, it is not known if the full-length protein (2551 residues) is even expressed as a stable product. We show in Zhou et al. (2003) that there are two isoforms of the human HARE protein and each isoform has discrete subunits. It is likely (but not yet confirmed) that all three of the large subunits in these two HARE isoforms are derived from the same initial gene product, as for the rat HARE (Zhou et al., 2002). Different posttranslational modifications, proteolytic processing, or multi-mer assemblies of the hypothetical 2551-amino-acid gene product could yield multiple discrete receptors with different ligand specificities. If so, then the structural differences among these species (e.g., N-terminal sequences or glycosylation patterns) will need to be elucidated. For example, a truncated portion of this gene product (the 175-kDa rat HARE isoform) is an endocytic HA receptor that functions via the coated pit pathway, even in the absence of the larger multiple-subunit isoform (Zhou et al., 2002).
In addition to multiple proteins, the field had other setbacks, such as the erroneous report that the endocytic HA receptor in liver was ICAM-1 (McCourt et al., 1994). Although the mistaken conclusion was later attributed to an artifact associated with the HA-affinity chromatography support used, this report confused the field for several years and was not formally withdrawn. Our concern about this study (and others cited by Smedsrod et al., 2003) is the lack of evidence of function for the candidate molecule, for example, a demonstration that cells transfected with cDNA encoding the candidate protein are then able to mediate endocytosis and degradation of HA or that specific monoclonal antibodies against the protein block HA receptor function in liver sinusoidal endothelial cells. To avoid similar problems in identifying the liver endocytic HA receptor, we provided both types of evidence for the molecular cloning of the small rat HARE isoform (Zhou et al., 2002). We reconstituted HA receptor function in transfected cells and developed a monoclonal antibody that blocks HA binding to the native and recombinant rat receptor. We submitted partial sequences for the HARE cDNAs to the USPTO on April 25, 2000.
Based on the use of a polycolonal antibody, McCourt et al. (1999) claimed that the liver sinusoidal endothelial cell receptor for HA and CS was also a scavenger receptor for AGE ligands, such as formaldehyde-treated bovine serum albumin and the N-terminal propeptide of collagen. Although this antibody also partially inhibited binding to the mannose receptor, they did not consider the clear implication that the antigen was not pure. We questioned the validity of the conclusions in this study; however, rather than criticize it, we chose not to cite it. Because few biochemical purification schemes ever yield a pure protein, the use of polyclonal antibodies prepared against such preparations is inherently unreliable for functional identification of a new protein. Discussion statements in Zhou et al. (2003) reflect the belief that many questions remain unanswered by this and the other studies cited by Smedsrod et al. (2003) regarding functional proof for identification of the protein responsible for HA receptor activity. This skepticism appears justified based on results reported by Hansen et al. (2002) demonstrating that the earlier conclusion about the remarkably broad ligand specificity of the HA-scavenger receptor was most likely incorrect. This study used rat liver sinusoidal endothelial cells to show that collagan, mannan, or HA does not inhibit the endocytosis of AGE–bovine serum albumin, formaldehyde-treated bovine serum albumin, or the collagen propeptide. That is, the HA receptor is apparently not a scavenger receptor for these latter three ligands—which result contradicts the conclusions of McCourt et al. (1999). The most likely explanation is that the polyclonal antibody used is not uniquely specific for the HA receptor but recognizes one or more scavenger receptors and the mannose receptor. A much less likely alternative explanation is that a single receptor has completely independent binding sites for the large polysaccharides HA, CS, and mannan; multiple AGE-related ligands; and collagen peptide. In any case, we believe that determining the correct explanation and proving the function of HARE, the HA-scavenger receptor or any other possible stabilin gene products will best be accomplished by the two approaches noted, using monoclonal or peptide-specific antibodies and well-characterized stable cell lines expressing the recombinant proteins.
References
Hansen, B., Arteta, B., and Smedsrod, B. (2002) The physiological scavenger receptor function of hepatic sinusoidal endothelial and Kupffer cells is independent of scavenger receptor class A type I and II. Mol. Cell. Biochem., 240, 1–8.[CrossRef][Web of Science][Medline]
Jackson, D.G., Prevo, R., Ni, J., and Ganerji, S. (2002) Novel endothelial hyaluronan receptors. In Kennedy, J.F., Philips, G.O.,and Williams, P.A. (Eds), Hyaluronan 2000. Woodhead Publishing, Wales, vol. 1, pp. 355–364.
McCourt, P.A., Ek, B., Forsberg, N., and Gustafson, S. (1994) Intercellular adhesion molecule-1 is a cell surface receptor for hyaluronan. J. Biol. Chem., 269, 30081–30084.
McCourt, P.A., Smedsrod, B.H., Melkko, J., and Johansson, S. (1999) Characterization of a hyaluronan receptor on rat sinusoidal liver endothelial cells and its functional relationship to scavenger receptors. Hepatology, 30, 1276–1286.[CrossRef][Web of Science][Medline]
Politz, O., Gratchev, A., McCourt, P.A., Schledzewski, K., Guillot, P., Johansson, S., Svineng, G., Franke, P., Kannicht, C., Kzhyshkowska, J., and others. (2002) Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues. Biochem. J., 362, 155–164.[CrossRef][Web of Science][Medline]
Smedsrod, B., Johansson, S., and Goerdt, S. (2003) Letter to the Glyco-Forum: shooting HARE. Glycobiology, 13, this issue.
Zhou, B., Oka, J.A., Singh, A., and Weigel, P.H. (1999) Purification and subunit characterization of the rat liver endocytic hyaluronan receptor. J. Biol. Chem., 274, 33831–33834.
Zhou, B., Weigel, J.A., Saxena, A., and Weigel, P.H. (2002) Molecular cloning and functional expression of the rat 175-kDa hyaluronan receptor for endocytosis. Mol. Biol. Cell, 13, 2853–2868.
Zhou, B., McGary, C.T., Weigel, J.A., Saxena, A., and Weigel, P.H. (2003) Purification and molecular identification of the human hyaluronan receptor for endocytosis (HARE). Glycobiology, 13, 339–349.
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