2 Department of Biochemistry and Molecular Biology, and The Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73190, USA
3 Department of Pathology, St Joseph's Hospital, 69 Exchange St., St. Paul, MN 55102, USA
Received on September 6, 2002; revised on November 6, 2002; accepted on November 6, 2002
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Abstract |
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Key words: hyaluronan binding protein / hyaluronic acid / iso-receptors / receptor-mediated endocytosis / recycling receptor
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Introduction |
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In previous studies using unique anti-rat monoclonal antibodies (mAbs), we purified this endocytic recycling HA receptor (Zhou et al., 1999), which has since (Zhou et al., 2000
) been designated the HA receptor for endocytosis (HARE), from rat liver LECs. We also demonstrated that HARE is localized to the LECs, as expected, but is even more abundantly expressed in the sinusoidal endothelial cells of the spleen and lymph nodes. We recently reported the first characterization of a functional cDNA for the rat 175-kDa HARE, confirming that this smaller HARE is an independent receptor able to function in the absence of the larger HARE species (Zhou et al., 2002
). Here we describe the first purification of the human HARE proteins using cross-reactive anti-rat HARE mAbs and identify the sequence of the human 190-kDa HARE protein.
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Results |
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When we tested the ability of our mAbs raised against the rat 175 kDa HARE to recognize a human HARE homolog, we found that three of the eight mAbs (numbers 30, 154, and 159) showed specific reactivity in western blots and immunocytochemistry. In particular, the level of expression of putative HARE proteins in human spleen was great enough to detect specific 125I-HA binding activity and western reactivity in crude extracts (data not shown).
The specific reactivity of the human HARE proteins with mAb 30, which had been used to purify the rat liver HARE, enabled us to purify HARE directly from detergent extracts of human spleen by immunoaffinity chomatography (Figure 1). Elution of proteins bound to mAb 30, using a low pH buffer, yielded the same two species identified in crude extracts as candidate HAREs. A 50-fold excess of nonlabeled HA inhibited 125I-HA binding to either of the two human HAREs by >90% (not shown). The two human HARE species at 190 kDa and
315 kDa are larger by about
15 kDa than the corresponding rat HARE species. HA-binding activity and reactivity with anti-rat HARE mAbs comigrated with these two major proteins at
190 kDa and
315 kDa. The anti-rat 175-kDa HARE mAbs that cross-react with the 190-kDa human HARE also recognize the
315-kDa HARE. Based on densitometric analysis of protein detected in western blots and ligand blots of the affinity purified proteins, the
315-kDa HARE is consistently more abundant that the 190-kDa HARE in human spleen. The apparent molar ratio of the
315-kDa HARE:190-kDa HARE in spleen is
23:1. Interestingly, an almost reverse ratio is observed for the two HARE iso-receptors in rat liver (Zhou et al., 1999
).
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Based on all of these results, we conclude that we have identified and assembled a partial human cDNA sequence for the full-length HARE, within which is encoded the 190-kDa HARE. The numbering convention for the 190 kDa HARE protein starts with the NH2-terminal Ser1 residue found in both spleen and lymph node cDNA pools (which were pooled from multiple individuals). NH2-terminal amino acid sequence analysis revealed a Leu residue at position 1 in the purified protein from one individual. When a nucleotide sequence error in BAB15793 (omission of an A, at position 695, which resulted in a frame shift) was corrected, the upstream ORF was continuous with our cDNA; this correction deleted eight amino acids at the N-terminal end of the ORF derived from BAB15793. A second error in the BAB-15793 nucleotide sequence at T1452 (rather than C) is silent.
The human core protein is smaller, at 1416 amino acids, than the corresponding rat protein (1431 amino acids), although the native human protein is larger as assessed by SDSPAGE (Figure 4, lanes 1 and 3). The possibility that carbohydrate modifications are responsible for the apparent size difference between the rat and human HARE proteins was confirmed by treatment of both proteins with endoglycosidase-F to remove N-linked oligosaccharides (Figure 4, lanes 2 and 4). The deglycosylated rat and human core proteins were virtually identical in size. Because the native human 190-kDa HARE is larger than the rat protein by 15 kDa, the human protein presumably has either more or larger N-linked oligosaccharides.
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The human 190-kDa HARE and the rat 175-kDa HARE protein sequences are 77% identical, with a gap frequency of only 0.2% (using the SIM Alignment Program), over a region containing 1416 residues (Figure 6). An additional 6.5% of the amino acid differences between the two proteins are conservative substitutions (e.g., R/K or S/T). Almost all of the cysteine residues within the extracellular domains of the two HARE proteins are absolutely conserved, which suggests that the two proteins have the same overall folding and organization of their polypeptide chains. The other HARE family members (Zhou et al., 2002) also share this extensive conservation of cysteine residues in their extracellular domains, as well as the same overall domain organization including the Xlink domain and a single predicted transmembrane region. Unlike the rat protein, the human HARE has no cysteine residues in its transmembrane or cytoplasmic domains. The cytoplasmic domains of the rat and human HARE proteins are less conserved (25% identical) than their transmembrane (76% identical) or extracellular domains (80% identical). Nonetheless, two candidate
XXB motifs for targeting these receptors to coated pits are highly conserved: the human HARE motifs YSYFRI1350 and FQHF1360 differ by only one amino acid from the corresponding regions in the rat HARE cytoplasmic domain (Figure 6).
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Discussion |
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The organization of the two HARE iso-receptors purified from human spleen is depicted in Figure 7. The 190-kDa and 315-kDa HAREs are most likely iso-receptors able to function independently as coated pit mediated endocytic receptors for HA. The 190-kDa HARE contains a single protein. The
315-kDa HARE is a disulfide-bonded complex that contains two to three copies of a 250-kDa subunit and one copy of a 220-kDa subunit. Spleen has about two to three times more of the
315-kDa HARE species compared with the 190-kDa HARE.
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The human HARE sequence reported here shares a high level of identity with the rat 175-kDa HARE and a family of human proteins (Zhou et al., 2002). One of these deduced human proteins, derived from accession number AAF82398, was designated FELL because it contains Fasciclin, EGF-like, and Link domains. The three sequences represented by AAF82398, CAB61358 and BAB15793, are
95% identical and may be the same sequence; the slight differences could be due to sequencing errors or alternative splicing. The sequences of CAB61827, which encodes Stabilin-1, and BAA13377 are more related to each other than to the three sequences noted or to HARE. Although the BAA13377 mRNA sequence is present in endothelial cells, the presence of protein or associated HA-binding activity was not determined (Tsifrina et al., 1999
). HARE and the BAB15793 sequence, which has recently been extended by several other submissions to the database, may be derived from a large protein originally designated Stabilin-2, deposited as a sequence of unconfirmed function (Politz et al., 2002
). The Stabilin-2 sequence was obtained from a soluble protein, which was isolated from whole liver extracts. Because we have confirmed the first function for a member of this protein family, it may now be more relevant to designate these proteins as HARE or HARE-like rather than as FELLs or Stabilins.
The overall similarities in their extracellular, transmembrane, and cytoplasmic domains suggest that the members of this HARE protein family may all be able to bind HA, chondroitin sulfate, or other glycosaminoglycans and mediate their endocytosis through the clathrin-coated pit pathway. The differences in their membrane and cytoplasmic domains also raise the possibility that the members of this family could interact with different membrane or cytoplasmic regulatory factors and, consequently, process or route these bound ligands through different intracellular pathways.
The human gene (stabilin 2) encoding HARE, which is in the genome database (originally under accession number NT_024383.2 and now NT_035234.1), is located on chromosome 12 and appears to be a highly interrupted gene. The nucleotide sequence representing the ORF for the portion that encodes the human 190-kDa HARE protein has over 40 exons interrupted by introns that vary in length from 143 to 70,255 nucleotides. The full-length HARE gene contains 69 exons that span 180.3 kb. All of the sequences within this region were deposited as putative or known gene products of unknown function. The present study provides the first identification of a function for the protein encoded by this gene. The human HARE nucleotide sequence reported here is >99% identical to the nucleotide sequence of the exons identified in the NT_024383.2 gene. The mouse HARE gene (accession number AC025501.3) has a similar organization, although the orientation and many of the HARE exons (assigned when the sequence was compiled) appear to be incorrect throughout this region of the gene.
Our current model for HA and chondroitin sulfate turnover in mammals highlights the role of HARE in liver and lymph nodes and to a lesser extent in spleen (Weigel and Yik, 2002). HARE mediates the uptake of HA into these tissues so it can be removed from the lymph, or blood, and degraded. A large fraction of the
5 g of HA turned over daily by humans is probably derived from skin, which contains about 50% of our total body HA (Laurent and Fraser, 1992
) and which, remarkably, has a half-life of only
11.5 day (Tammi et al., 1991
). Presently, there are important clinical uses for HA-containing devices in treating wounds, osteoarthritis, and in eye surgery (Abatangelo and Weigel, 2000
; Panay and Lower, 1999
).
Additional future uses of HA in clinical applications are likely to be developed based on our growing understanding of the biology of HA and its multiple roles in wound healing (Iocono et al., 1998; Chen and Abatangelo, 1999
), angiogenesis (West et al., 1985
; Deed et al., 1997
; Rahmanian et al., 1997
), macrophage activation (Horton et al., 1998
, 2000
), and metastasis (Csoka et al., 1997
; Delpech et al., 1997
). A variety of different drug delivery systems utilizing HA are also being developed (Cantor et al., 1998
; Illum et al., 1994
; Luo et al., 2000
). Given the likely increase in the clinical uses of HA-containing devices and drugs, it is now important that we understand the overall mechanism of HA turnover in the body. In particular, the present molecular identification and characterization of the human receptor responsible for HA clearance is timely and should facilitate further studies in this field.
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Materials and methods |
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Purification and sequence analysis of the HARE from human spleen
Human spleen tissue was obtained from a 14-year-old female patient undergoing splenectomy for hereditary spherocytosis following approval from the University of Rochester Research Subjects Review Board. The tissue was cut into small pieces on ice, added to 2% Nonidet P-40 in TBS containing a mixture of proteinase inhibitors (2 mM diisopropyl fluorophosphate, 1 mM phenylmethylsulfonyl fluoride, and 1 mM N-ethylmaleimide), then homogenized using a Tissumizer power homogenizer (Tekmar, Cincinnati, OH) and incubated at 4°C for 1 h. The extract was diluted fourfold with TBS and centrifuged at 12,000 x g for 30 min at 4°C. The supernatant was loaded onto a column containing anti-rat HARE mAb-30 (Zhou et al., 1999, 2000
) for affinity chromatography (
2 mg/ml IgG coupled to CNBr-activated Sepharose). The same antibody was used previously to purify the rat HARE (Zhou et al., 1999
). The column was washed with 10 volumes of TBST and then eluted with 100 mM sodium citrate, pH 2.5. Eluted fractions were neutralized with 1 M Tris, pooled, dialyzed against TBS at 4°C, and concentrated using a Centricon-30 device (Amicon, part of Millipore, Bedford, MA).
The concentrated sample was subjected to SDSPAGE and in some cases then transferred to nitrocellulose. The HA-binding activity of HARE was determined by a 125I-HA ligand-blot assay, and HARE protein was localized by western blot analysis as will be described. Purified human HARE preparations were subjected to SDSPAGE, and gels were stained with Coomassie blue to identify the proteins. The 190-kDa human HARE protein band was excised and sent to the Protein Chemistry Lab at the University of Texas Medical Branch at Galveston for trypsin digestion and amino acid sequence analysis of internal peptides. Samples of the tryptic digests were also sent to the Mass Spectroscopy Facility at Louisiana State University.
Purification of human spleen mRNA and RT-PCR
Human spleen tissue was cut into small pieces and homogenized on ice using TRIzol reagent to isolate the total RNA. The mRNA was isolated from total RNA using a PolyATtract mRNA Isolation Kit following the manufacturer's recommended protocols. First-strand cDNA was synthesized using the Thermoscript II RT-PCR system from Life Technologies (part of Invitrogen, Carlsbad, CA) with random hexameric oligonucleotides or oligo(dt)20. The PCR reactions using Advantage 2 cDNA Polymerase Mix from Clontech were carried out with incubation at 94°C for 2.5 min; 35 cycles of 50°C for 1 min, 68°C for 3 min, and 94°C for 1 min; and one cycle of 50°C for 1 min and 68°C for 15 min. Oligonucleotide primers were based on either the human 190-kDa HARE peptide sequences (HSP primers) or the nucleic acid sequence under GenBank accession number BAB15793 (BAB primers), which is derived from accession number AK024503 (i.e., the mRNA for FLJ00112 protein). PCR products were obtained using the following pairs of oligonucleotides (Table I): BAB1F-HSP2R, HSP3F-BAB4R, BAB6F-BAB7R, BAB1F-BAB10R, HSP2F-BAB10R, BAB10F-HSP3R, and BAB9F-HSP3R. The PCR products were subjected to electrophoresis using a 1% (w/v) agarose gel, and the DNA bands were excised and purified using a Gel Extraction Kit. The purified PCR DNA products were cloned into pCR4-TOPO vector using the TOPO-TA Cloning Kit (Invitrogen, Carlsbad, CA). The colonies were screened by PCR, and the DNA insert size was verified by restriction enzyme digestion using EcoRI. The plasmid DNAs from positive clones were purified using QIAprep Spin Plasmid Kits, and the complete inserts were sequenced by the DNA Sequencing Facility of the Oklahoma Medical Research Foundation, Oklahoma City, OK.
Ligand blot assay
Purified human spleen HARE preparations were mixed with equal volumes of a 2x SDS sample buffer (Laemmli, 1970) to give final concentrations of 16 mM TrisHCl, pH 6.8, 2% (w/v) SDS, 5% glycerol (v/v), and 0.01% bromophenol blue. No reducing agent was added unless as noted in the figures. After SDSPAGE, the contents of the gel were electrotransferred to a nitrocellulose membrane for 2 h at 24 V at 4°C using 25 mM Tris, pH 8.3, 192 mM glycine, 20% methanol, and 0.01% SDS in a Genie blotter apparatus from Idea Scientific (Minneapolis, MN). The nitrocellulose membrane was treated with TBS containing 0.1% Tween-20 at 4°C for 2 h or TBST overnight and then incubated with 2 µg/ml 125I-HA in TBS without or with a 150-fold excess of nonlabeled HA (as competitor) to assess total and nonspecific binding, respectively (Yannariello-Brown et al., 1996
, 1997
). The nitrocellulose membrane was washed five times for 5 min each with TBST and dried at room temperature. The bound 125I-HA was detected by autoradiography with Kodak (Rochester, NY) BioMax film. Nonspecific binding in this assay is typically <5%.
Western blot analysis of human HARE
Nitrocellulose membranes were blocked with 1% bovine serum albumin in TBS at 4°C overnight either after the ligand blot assay or directly after SDSPAGE and electrotransfer (Burnette, 1981) as described. The membrane was then incubated with anti-rat HARE mAbs (e.g., 1:5000 dilution of ascites) at room temperature for 2 h, washed five times for 5 min each with TBST, and incubated with goat anti-mouse IgG conjugated to alkaline phosphatase for 1 h at room temperature. The nitrocellulose was washed five times for 5 min each with TBST and incubated with the substrates p-nitro blue tetrazolium and sodium 5-bromo-4-chloro-3-indolyl phosphate p-toluidine for color development (Bio-Rad), which was stopped by washing the membrane with distilled water.
Two-dimensional electrophoresis of human HARE
Immunoaffinity-purified human HARE was first subjected to SDSPAGE without reduction and the gel was stained with Coomassie blue. The 190-kDa and 315 kDa-HARE protein bands were excised from the gel, cut into small pieces, divided into two portions, and then incubated in SDS sample buffer with or without 1.25% ß-mercaptoethanol at 90°C for 4 min; identical results were also obtained with or without reduction using 10 mM dithiothreitol followed by alkylation with 50 mM iodoacetamide. The samples were then subjected to a second dimension of SDSPAGE in the absence of reducing agent and parallel portions were processed in gel to identify the proteins by silver staining (Blum et al., 1987
).
Immunocytochemistry
Human lymph node, liver, and spleen tissues were identified by search of the computerized surgical pathology database at the University of Rochester following approval from the Institutional Research Subjects Review Board. The original hematoxylin and eosin-stained sections were reviewed, and appropriate tissue blocks were selected for staining. The tissues had been fixed in 10% neutral buffered formalin at the time of surgery, processed, and paraffin embedded overnight on a Tissue Tek V.I.P. processor. Tissue sections (5 µm) were collected on charged slides and dried at 60°C overnight. The slides were dewaxed three times for 3 min each with xylene, followed by four washes for 3 min each with alcohol (100%, 95%, 90%, 70%), followed by a single 2-min wash in water at room temperature.
The endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 6 min, followed by two 2-min water washes. The slides were digested for 15 min at 37°C in prewarmed 0.1 N HCl containing 0.32 mg/ml pepsin, followed by a 2-min water wash and a 2-min phosphate buffered saline (PBS) wash. The slides were washed with PBS and incubated with the appropriate primary antibody (1:500) at room temperature for 60 min. After a 1-min PBS wash, the slides were treated with secondary antiserum (biotinylated horse anti-mouse, 1:200) for 30 min at room temperature. After another PBS rinse, the slides were incubated with streptavidinhorseradish peroxidase (1:1000 from Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min, washed once with PBS, and washed once with distilled water. Color development was for 5 min with 2.0% (v/v) aminoethylcarbazine and hydrogen peroxide according to the manufacturer's instructions (ScyTek, UT), followed by counterstaining with hematoxylin. Slides were viewed with an Olympus BX-40 light microscope equipped with an Olympus DP10 digital camera for photography.
General
Protein content was determined by the method of Bradford (1976) using bovine serum albumin as a standard. SDSPAGE was performed according to the method of Laemmli (1970)
. 125I radioactivity was measured using a Packard 5002 Auto-Gamma Counting system.
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Acknowledgements |
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1 To whom correspondence should be addressed; e-mail: paul-weigel{at}ouhsc.edu
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Abbreviations |
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References |
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