Purification and molecular identification of the human hyaluronan receptor for endocytosis

Bin Zhou2, Carl T. McGary3, Janet A. Weigel2, Amit Saxena2 and Paul H. Weigel1,2

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


    Abstract
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of ~175 kDa and ~300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733–37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS–PAGE, these two human HARE species migrated at ~190 kDa and ~315 kDa; both proteins are ~15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the ~315-kDa HARE after reduction. The reduced ~315-kDa HARE yielded two major proteins at ~250 kDa and ~220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.

Key words: hyaluronan binding protein / hyaluronic acid / iso-receptors / receptor-mediated endocytosis / recycling receptor


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The synthesis and degradation of hyaluronan (HA) is important for many aspects of cell behavior and function. HA is a high-molecular-weight linear polysaccharide found in the extracellular matrix of all vertebrate tissues. The polysaccharide is an alternating copolymer composed of only two sugars: ß(1,3)-linked-D-glucuronic acid and ß(1,4)-linked-N-acetyl-D-glucosamine. A wide range of cellular activities utilize or require HA, including cell proliferation, cell adhesion, recognition, morphogenesis, differentiation, and inflammation (Laurent and Fraser, 1992Go; Knudson and Knudson, 1993Go; Toole, 1997Go; Abatangelo and Weigel, 2000Go). Every day in tissues throughout our bodies, we continuously synthesize and degrade as much as 5 g of HA (Laurent and Fraser, 1991Go). Because the total body content of HA in a typical 70-kg adult is ~15 g, we therefore turn over about one-third of our total body HA every day. Laurent and Fraser (1992)Go and their co-workers showed that this large burden of HA turnover in mammals is mediated primarily by the liver and lymph nodes. This group and ours demonstrated that the sinusoidal liver endothelial cells (LECs) have a very active, recycling, endocytic receptor that removes HA and a variety of chondroitin sulfates from the circulation (Fraser et al., 1983Go, 1985Go; Laurent et al., 1986Go; Raja et al., 1988Go; McGary et al., 1989Go). Presumably, the removal of this circulating HA is necessary for normal health. Because the rate of HA turnover is so high, failure to remove it would likely lead to extremely elevated concentrations of HA in the blood within days. The physiological consequences of increased blood viscosity due to accumulation of HA are not known, but elevated serum HA levels are found in a variety of disease conditions such as liver cirrhosis, rheumatoid arthritis, psoriasis, scleroderma, and some cancers (Lai et al., 1998Go; Manicourt et al., 1999Go; Freitas et al., 1996Go; Thylen et al., 1999Go).

In previous studies using unique anti-rat monoclonal antibodies (mAbs), we purified this endocytic recycling HA receptor (Zhou et al., 1999Go), which has since (Zhou et al., 2000Go) 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., 2002Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Identification and purification of human HARE
We used a specific mAb against the rat 175-kDa HARE to purify the two rat liver HARE proteins (Zhou et al., 1999Go) that had been identified in earlier studies employing a photoaffinity derivative of HA (Yannariello-Brown et al., 1992Go) and a novel ligand blot assay (Yannariello-Brown et al., 1996Go, 1997Go). Zhou et al. (2002)Go showed that the 175-kDa HARE is functional as an endocytic receptor for HA in the absence of the 300-kDa HARE; therefore, these two HARE species are iso-receptors for HA. The rat 175-kDa and ~300-kDa HARE proteins are each able to bind 125I-HA in a ligand blot assay following nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and electrotransfer. Furthermore, they are immunologically related, because all mAbs raised against the 175-kDa HARE also recognize the 300-kDa HARE (Zhou et al., 2000Go). The 175-kDa rat HARE contains only one protein, whereas the rat 300-kDa HARE contains three distinct disulfide-bonded subunits of about 260 kDa, 230 kDa, and 97 kDa (Zhou et al., 1999Go). The 260-kDa and 230-kDa subunits of the 300-kDa HARE are both recognized by the panel of anti-175-kDa HARE mAbs.

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 ~2–3:1. Interestingly, an almost reverse ratio is observed for the two HARE iso-receptors in rat liver (Zhou et al., 1999Go).



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Fig. 1. Immunoaffinity purification of the human HARE proteins. Human HAREs were purified from Nonidet P-40 extracts of human spleen tissue by affinity chromatography using the anti-rat 175-kDa HARE mAb-30. The HARE proteins were eluted at low pH, concentrated, subjected to SDS–PAGE, and transferred to nitrocellulose. The membrane was stained with 0.05% copper phthalocyanine tetrasulfonic acid, tetrasodium salt (lane 1) and then destained with distilled water. The destained nitrocellulose membrane was treated with TBST to block nonspecific binding sites and assessed for 125I-HA-binding activity (lane 2). After the ligand blot assay, the nitrocellulose membrane was subjected to western blot analysis using a mixture of mAbs raised against the rat 175-kDa HARE protein (lane 3). The open and closed arrows indicate the positions of the human HARE proteins at ~190 kDa and ~315 kDa, respectively. These two human HARE species correspond to the previously characterized rat 175-kDa and 300-kDa HARE proteins (Zhou et al., 1999Go).

 
Subunit characterization of the two human HARE iso-receptors
The affinity purified 190-kDa HARE and ~315-kDa HARE proteins were subjected to two-dimensional SDS–PAGE analysis to determine their subunit compositions (Figure 2). After reduction, the 190-kDa HARE yielded a single major protein that migrated at ~196 kDa, indicating that the smaller HARE contains only one polypeptide. Two smaller minor bands are likely degradation products of the larger protein. As with the rat 175-kDa HARE (Zhou et al., 1999Go), the reduced protein is unfolded and thus migrates more slowly in SDS–PAGE. This behavior is typical for receptors with extracellular domains containing disulfide bonds, such as the Cys-rich HARE proteins (Zhou et al., 2002Go). The ~315-kDa HARE yielded two major proteins of ~220 and ~250 kDa after reduction, indicating that the larger HARE contains at least two types of disulfide-bonded subunits. We have consistently observed these two subunits in multiple independent preparations of purified human spleen HARE. The apparent molar ratio of the 250 kDa:220 kDa subunits is about 2–3:1. In contrast, the rat 300-kDa HARE contains three subunits in apparent molar ratios of 1:1:1, respectively (Zhou et al., 1999Go).



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Fig. 2. Subunit analysis of the purified human 190-kDa and ~315-kDa HARE proteins. The two human HARE proteins were immunoaffinity-purified from human spleen using anti-rat 175 HARE mAb-30, subjected to nonreducing SDS–PAGE, and the gels were stained with Coomassie blue (lane 1) to visualize the HARE proteins. The 190-kDa HARE and ~315-kDa HARE protein bands were excised, minced, and divided into two portions for analysis by SDS–PAGE, with or without reduction using ß-mercaptoethanol, followed by silver staining. The 190-kDa HARE protein gives a single ~196-kDa species either with (lane 4) or without (lane 2) reduction. The excised 315-kDa HARE is a single band without reduction (lane 3) but gives two major species after reduction (lane 5), one at ~250 kDa and another at ~220 kDa.

 
Localization of HARE in human tissues
Using mAb 30, which works well in immunocytochemical applications, we found abundant HARE protein expression in human liver, spleen, and lymph node (Figure 3). Staining intensity and therefore protein expression levels were much greater in lymph node than in spleen or liver. In each tissue, only cells in the sinusoidal regions were stained. In spleen, the germinal centers and white pulp areas of splenic nodules were unstained, whereas the venous sinusoids of the red pulp stained strongly (Figure 3A). Controls using mouse serum showed very low background staining in spleen (Figure 3B) or the other tissues (not shown).



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Fig. 3. Immunocytochemical localization of HARE in human liver, spleen, and lymph node. Sections of human spleen (A and B), lymph node (C), and liver (D) were treated with anti-HARE mAb-30 (A, C, and D) or mouse serum (B) and then stained as described in Materials and methods. A relatively low magnification is shown (the bar represents ~500 µm) to emphasize the localization of the human HARE protein in the sinusoidal regions of each tissue.

 
Analysis of NH2-terminal and internal peptide sequences and determination of the 190-kDa HARE sequence
The affinity-purified HARE proteins were reduced and separated by SDS–PAGE; the band corresponding to the reduced 190-kDa HARE was excised and subjected to internal peptide analysis following trypsin digestion. When the protein databases were searched using the amino acid sequences derived from the purified protein, an identical match was found for a subset of seven peptides (Table I) predicted to be within a hypothetical human protein of unknown function under accession number BAB15793. Seven additional tryptic peptides derived from the 190-kDa HARE were matched by mass spectroscopic analysis (within <0.2 Da) to the same deduced sequence in the database (Table II). This sequence had also been independently identified (Zhou et al., 2002Go) as the most likely human homolog of HARE based on an overall homology of ~85% (78% identity) between the 1431-amino-acid rat 175-kDa HARE and a putative 1193-amino-acid protein encoded by BAB15793. The purified protein was also subjected to automated amino acid sequence analysis, and the NH2-terminal sequence was identified as XLLPNLLMRL. This latter sequence is 67% identical to the NH2-terminus of the rat 175-kDa HARE and was not identified in the putative open reading frame (ORF) encoded by BAB15793, as will be discussed.


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Table I. Summary of amino acid sequences derived from peptides of the purified human 190 kDa HARE protein

 

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Table II. Molecular mass mapping of peptides derived from the human 190-kDa HARE protein

 
The identical match of 14 tryptic peptides that contain a total of 120 amino acids (~10% of the protein), including a 23-residue peptide (Table I), should be enough to conclude that the partial sequence encoded by BAB15793 is part of the human HARE. To support this conclusion further, however, we utilized reverse transcriptase polymerase chain reaction (RT-PCR) with human spleen or lymph node mRNA and a combination of human HARE-specific and BAB15793-specific primers (BABs) to identify, clone, and sequence PCR products that span portions of a ~4-kb region of the HARE-coding sequence (Table III). The eight PCR products shown in Table III each encoded two to four tryptic peptides predicted to be in the BAB15793 protein and confirm the relationship between the purified human spleen HARE and the partial protein sequence deduced from BAB15793.


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Table III. Oligonucleotide primer pairs used for RT-PCR analysis and DNA sequencing

 
The nucleic acid sequence and deduced protein sequence for the 190 kDa HARE are deposited under GenBank accession number AY227444. The BAB15793 nucleotide sequence contains a partial ORF of 1193 amino acids that starts at nucleotide position 606. Our RT-PCR products generated from spleen mRNA confirmed almost all of the 4575-bp BAB15793 sequence with several important exceptions. Most significant, key results characterizing new human HARE sequences were obtained from the most 5' RT-PCR product generated from primer pair BAB1F (derived from an upstream region of BAB15793 that had been incorrectly identified as untranslated) and HARE-specific primer (HSP) 2R. The majority of this 420-bp PCR product is upstream of the putative Trp residue (see figure 12 in Zhou et al., 2002Go) that begins the BAB15793 hypothetical protein sequence. In fact, the first seven residues of this hypothetical sequence were incorrect due to a frame shift error. The 140 amino acid sequence derived from the 1F–2R PCR product is in frame with and extends the size of the human HARE ORF. This partial ORF is at least 4182 bp, ends at a stop codon, and encodes a protein of 1394 residues. This cDNA was further extended by 5' rapid amplification of cDNA ends to a position equivalent to the NH2 terminus of the rat 175-kDa HARE. We also obtained an additional upstream in-frame sequence encoding 227 residues. Only a few minor nucleotide differences were found between the spleen and lymph node cDNAs for HARE.

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 SDS–PAGE (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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Fig. 4. Deglycosylation of HARE. One microgram of purified rat (lanes 1 and 2) and human (lanes 3 and 4) HARE proteins were denatured by boiling in 0.5% SDS, mixed with 0.5% NP-40 and de-N-glycosylated using ~5 units N-glycosidase F at 37°C overnight. After SDS–PAGE and electrotransfer to nitrocellulose, the HARE protein bands were detected using a mixture of anti-HARE mAbs.

 
Domain organization and characteristics of the 190-kDa HARE
The overall organization of the human 190-kDa HARE protein is essentially identical to that of the rat 175-kDa HARE (Zhou et al., 2002Go). The human protein is predicted to be a type I membrane protein (Figure 5), with a large NH2-terminal extracellular domain (~1324 amino acids), a single transmembrane domain (~21 amino acids), and a small COOH-terminal cytoplasmic domain (~72 amino acids). The predicted mass of the 1416-residue core protein is 154,090 Da and the pI is pH 5.9.



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Fig. 5. Domain organization of the human 190-kDa HARE. The scheme depicts the organization of protein domains identified by the programs Pfam-HMM, CD-Search, ScanProsite, or SMART (Schultz et al., 1998Go). Abbreviations used are TMD (transmembrane domain) and EGF (epidermal growth factor). Potential N-linked oligosaccharides at -N-X-T/S- sites are indicated by the carets.

 
The protein contains 17 potential N-glycosylation sites (-N-X-T/S-) in the extracellular domain. Twelve of these sites are identical with sites in the rat 175-kDa HARE (Figure 6). An additional three nonclassical glycosylation sequons (-N-X-C-) are present in the human HARE, two of which are conserved with the rat HARE. An interesting feature of such Cys-containing sites is that glycosylation and participation of the Cys in a disulfide bond may be mutually exclusive (Miletich and Broze, 1990Go). The 190-kDa HARE extracellular domain has a delta serrate ligand domain, and four ß-Ig-H3/fasciclin-like domains,three metallothionein domains, four furin-like domains, a Link domain, and ~24 epidermal growth factor (EGF)-like domains (many of which overlap) arranged in two cysteine-rich clusters separated by a 353-amino-acid region that is cysteine-poor, as well as one 93-amino-acid Link (or Xlink) domain near the membrane junction (Gly1063–Tyr1155). Many of the programs, such as Pfam-HMM, ScanProsite, SMART (Schultz et al., 1998Go), or CD-Search, identify domains that are only partial or weak matches (e.g., early protein and delta serrate ligand domains) or overlap with other domains. In particular, the EGF-like domains (Figure 5) show this latter characteristic. Although the overall organization of all of these domains is very similar between the human and rat HARE proteins, the exact arrangement and number of each type of domain is not identical.



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Fig. 6. Sequence alignment of the human and rat HARE proteins. Sequences for the two smaller HARE proteins were aligned using SIM (at www.ExPASy) and then annotated in Microsoft Word. Identical residues found in both sequences are noted with dashes in the rHARE sequence. Fourteen conserved consensus N-linked glycosylation sites are in boldface and underlined, including two potential -N-X-C- glycosylation sites. Cys residues identical between the two proteins are in boldface with light gray highlighting. Two gaps in the rat sequence are noted by equal signs. The arrow denotes the least conserved regions of the two proteins, which are in their cytoplasmic domains. The residues under the solid line are an extracellular Link domain (Xlink), a putative hyaluronan-binding domain. Residues under the dashed line indicate the single predicted transmembrane domain. The three conserved candidate {Phi}XXB motifs are within the two boxes. Ser, Thr, or Tyr residues predicted (by NetPhos 2.0) to be phosphorylated are shown in boldface white with black highlighting. Our deposited sequence for the rat 175 kDa HARE is under accession numbers AY007370 and AAG13634 for the nucleic acid and protein sequences, respectively. The human 190 kDa HARE nucleic acid sequence is under GenBank accession number AY227444. The human 190 kDa HARE protein contains 1416 amino acids.

 
The cytoplasmic domain of the human HARE (~Y1345-L1416) contains four Tyr, seven Ser, one His, and five Thr residues, although only residues S1362, S1402, T1388, Y1384, and Y1396 are predicted (by NetPhos 2.0) to be phosphorylated. PEST motifs for rapid degradation or consensus sequences for O-glycosylation with GlcNAc are not present. As expected, the cytoplasmic domain contains several putative candidate motifs for targeting the protein to clathrin-coated pits. The sequence YSYFRI1350 at the junction between the transmembrane and cytoplasmic domains contains an interesting overlapping combination of two {Phi}XXB motifs, where {Phi} is either tyrosine or phenylalanine, X can be any amino acid, and B is a hydrophobic residue with a bulky side chain.

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., 2002Go) 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 {Phi}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).


    Discussion
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
The rat 175-kDa HARE protein is a functional endocytic HA receptor when expressed from a nonnaturally occurring synthetic cDNA (Zhou et al., 2002Go). The protein is not directly encoded by an mRNA but is derived from the proteolytic processing of a larger protein, which may be the large 260-kDa subunit of the 300-kDa HARE complex (Zhou et al., 1999Go). The mRNA encoding the rat 175-kDa HARE is ~10 kb, substantially longer than that required for this size protein. The strikingly similar characteristics of the rat and human HAREs indicate that a similar proteolytic processing generates the human 190-kDa HARE from one of the large subunits of the 315-kDa HARE. The two human HARE iso-receptors described here have a very similar organization to the two rat HAREs, and the three anti-rat mAbs that recognize the 190-kDa human HARE also cross-react with the two large subunits of the human ~315-kDa HARE.

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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Fig. 7. Model for the organization of the two human spleen HARE iso-receptors. The 190-kDa and ~315-kDa HARE iso-receptors isolated from spleen are depicted as separate species in approximate molar ratios of 1:2, respectively. The 190-kDa HARE contains only one protein. The large HARE complex is composed of two (or perhaps three) disulfide-bonded subunits of about 250 kDa and one subunit of 220 kDa. The molar ratios of the two HARE iso-receptors may be different in different tissues. All HARE proteins and subunits are membrane-bound and are predicted to contain small cytoplasmic domains and very large ectodomains. The HARE proteins are elongated rather than globular (Yannariello-Brown et al., 1997Go).

 
The Link domain is clearly a good candidate for an HA-binding region, but it is very likely that other, perhaps multiple, non-Link HA-binding domains are also present in the extracellular domain of HARE. Day, Jackson, and colleagues have extensively investigated the structural requirements for HA-binding activity of Link domains from different proteins (Bajorath et al., 1998Go; Kahmann et al., 2000Go; Banerji et al., 1998Go; Mahoney et al., 2001Go). In general, the affinity constants of these link domains are in the 106 M-1 range, which is not suitable for efficient receptor-mediated endocytosis. Receptor–ligand complexes targeted to coated pits typically have Kd values in the 1–50 nM range. The longer ~315-kDa HARE iso-receptor probably has more HA-binding domains than the smaller 190-kDa HARE.

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., 2002Go). 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., 1999Go). 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., 2002Go). 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, 2002Go). 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, 1992Go) and which, remarkably, has a half-life of only ~1–1.5 day (Tammi et al., 1991Go). Presently, there are important clinical uses for HA-containing devices in treating wounds, osteoarthritis, and in eye surgery (Abatangelo and Weigel, 2000Go; Panay and Lower, 1999Go).

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., 1998Go; Chen and Abatangelo, 1999Go), angiogenesis (West et al., 1985Go; Deed et al., 1997Go; Rahmanian et al., 1997Go), macrophage activation (Horton et al., 1998Go, 2000Go), and metastasis (Csoka et al., 1997Go; Delpech et al., 1997Go). A variety of different drug delivery systems utilizing HA are also being developed (Cantor et al., 1998Go; Illum et al., 1994Go; Luo et al., 2000Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
Materials
Copper phthalocyanine tetrasulfonic acid tetrasodium salt was obtained from Aldrich Chemical (Milwaukee, WI). Tris, SDS, ammonium persulfate, N,N'-methylenebisacrylamide, and SDS–PAGE molecular weight standards were from Bio-Rad (Hercules,CA). N-glycosidase F and Nonidet P-40 were from Calbiochem (Darmstadt, Germany). Na125I was from Amersham. 1,3,4,6-Tetrachloro-3{alpha}, 6{alpha}-diphenylglycouril was from Pierce (Rockford, IL). HA (human umbilical cord) obtained from Sigma (St. Louis, MO), was further purified as described previously by fractionation on celite and ethanol precipitation (Raja et al., 1984Go). 125I-HA was prepared as described previously (Raja et al., 1984Go), using a derivative of HA uniquely modified at the reducing end to contain a hydroxyphenylpropionylamidohexyl group and purified by chromatography over a PD-10 column (Amersham, Piscataway, NJ). Nitrocellulose membranes (0.1 µm pore size) were from Schleicher & Schuell (Keene, NH). Acrylamide and urea were from U.S. Biochemical (Cleveland, OH). Marathon human spleen and lymph node cDNA pools were obtained from Clontech (part of BD Biosciences, Palo Alto, CA). All other chemicals were reagent grade and were from Sigma. Tris buffered saline (TBS) contains 20 mM Tris–HCl, pH 7.0, 150 mM NaCl. TBST is TBS containing 0.05% Tween 20 and 0.05% NaN3.

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., 1999Go, 2000Go) 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., 1999Go). 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 SDS–PAGE 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 SDS–PAGE, 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, 1970Go) to give final concentrations of 16 mM Tris–HCl, 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 SDS–PAGE, 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., 1996Go, 1997Go). 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 SDS–PAGE and electrotransfer (Burnette, 1981Go) 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 SDS–PAGE 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 SDS–PAGE in the absence of reducing agent and parallel portions were processed in gel to identify the proteins by silver staining (Blum et al., 1987Go).

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 streptavidin–horseradish 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)Go using bovine serum albumin as a standard. SDS–PAGE was performed according to the method of Laemmli (1970)Go. 125I radioactivity was measured using a Packard 5002 Auto-Gamma Counting system.


    Acknowledgements
 
We thank Steve Smith and Dr. Alex Kurosky at the University of Texas Medical Branch at Galveston for internal peptide analyses; Robert Raymond and Dr. Ed Harris for performing the NH2-terminal sequence analysis; Anil Singh for technical assistance; and Sheryl Christofferson of the Oklahoma Medical Research Foundation DNA Sequencing Facility. This research was supported by NIH grant GM35978 from the National Institute of General Medical Sciences. The nucleic acid and protein sequences of human HARE reported here are in the GenBank database under accession number AY22744.

1 To whom correspondence should be addressed; e-mail: paul-weigel{at}ouhsc.edu Back


    Abbreviations
 
BAB, BAB15793-specific primer; EGF, epidermal growth factor; FELL, Fasciclin, EGF-like, and Link; HA, hyaluronic acid, hyaluronate, hyaluronan; HARE, HA receptor for endocytosis; HSP, HARE-specific primer; LECs, sinusoidal liver endothelial cells; mAb, monoclonal antibody; ORF, open reading frame; PBS, phosphate buffered saline; RT-PCR, reverse transcriptase polymerase chain reaction; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TBST, TBS containing 0.05% Tween 20


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 References
 
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