Molecular Heterogeneity of the SHAP-Hyaluronan Complex
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m303658200
ISSN1083-351X
AutoresWannarat Yingsung, Lisheng Zhuo, Matthias Mörgelin, Masahiko Yoneda, Daihei Kida, Hideto Watanabe, Naoki Ishiguro, Hisashi Iwata, Koji Kimata,
Tópico(s)Platelet Disorders and Treatments
ResumoWe previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-α-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAP-to-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 × 106 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex. We previously found that a covalent complex of SHAPs (serum-derived hyaluronan-associated proteins), the heavy chains of inter-α-trypsin inhibitor family molecules, with hyaluronan (HA) is accumulated in synovial fluid of patients with rheumatoid arthritis, and the complex is circulated in patient plasma at high concentrations. How the SHAP-HA complex participates in this disease is unknown. To address this question, it is essential to clarify the structural features of this macromolecule. The SHAP-HA complex purified from synovial fluid of the patients by three sequential CsCl isopycnic centrifugations was heterogeneous in density, and the fractions with different densities had distinct SHAP-to-HA ratios. Agarose gel electrophoresis and column chromatography revealed that there was no apparent difference in the size distribution of HA to which SHAPs were bound between the fractions with different densities. The SHAP-HA complex in the higher density fraction had fewer SHAP molecules per HA chain. Therefore, the difference between the fractions with different densities was due to a heterogeneous population of the SHAP-HA complex, namely the different number of SHAP molecules bound to an HA chain. Based on the SHAP and HA contents of the purified preparations, we estimated that an HA chain with a molecular weight of 2 × 106 has as many as five covalently bound SHAPs, which could give a proteinaceous multivalency to HA. Furthermore, we also found that the SHAP-HA complex tends to form aggregates, judging from the migration and elution profiles in agarose gel electrophoresis and gel filtration, respectively. The multivalent feature of the SHAP-HA complex was also confirmed by the negative staining electron micrographic images of the purified fractions. Taken together, those structural characteristics may underlie the aggregate-forming and extracellular matrix-stabilizing ability of the SHAP-HA complex. Hyaluronan (HA), 1The abbreviations used are: HA, hyaluronan; SHAPs, serum-derived hyaluronan-associated proteins; HC, heavy chain; ITI, inter-α-trypsin inhibitor; PαI, pre-α-trypsin inhibitor; HABP, hyaluronan-binding protein; CB, Coomassie Brilliant Blue; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; HRP, horseradish peroxidase; BSA, bovine serum albumin; TMB, The 3, 3′, 5, 5′-tetramethylbenzidine.1The abbreviations used are: HA, hyaluronan; SHAPs, serum-derived hyaluronan-associated proteins; HC, heavy chain; ITI, inter-α-trypsin inhibitor; PαI, pre-α-trypsin inhibitor; HABP, hyaluronan-binding protein; CB, Coomassie Brilliant Blue; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; HRP, horseradish peroxidase; BSA, bovine serum albumin; TMB, The 3, 3′, 5, 5′-tetramethylbenzidine. a high molecular weight linear glycosaminoglycan composed of repeating disaccharide units of glucuronosyl-N-acetylglucosamine, distributes ubiquitously in most mammalian connective tissues and the body fluids (1Balaz E.A. Balazs E.A. Jeanloz R.W. The Amino Sugar. Vol. IIA. Academic Press, New, York1965: 401-460Google Scholar, 2Sundblad L. Balazs E.A. Jeanloz R.W. The Amino Sugar. Vol. IIA. Academic Press, New, York1965: 250-299Google Scholar, 3Laurent T.C. Fraser J.R. FASEB J. 1992; 6: 2397-2404Crossref PubMed Scopus (2041) Google Scholar). The association with various hyaluronan-binding proteins (HABPs) including proteoglycans makes HA tremendously divergent in physiological function. For example, link proteins and aggrecan, major HABPs in cartilage, are implicated in the characteristic functions of this tissue such as elasticity by forming large macromolecular aggregates with HA in the extracellular matrix. Such extracellular matrices containing HA as a major component, which, hereafter, we call HA-rich matrices, play important roles in regulating cellular behavior in a variety of physiological and pathological processes via cell surface HA receptors, such as CD44 and RHAMM (4Toole B.P. Curr. Opin. Cell Biol. 1990; 2: 839-844Crossref PubMed Scopus (382) Google Scholar, 5Heinegård D. Björnsson S. Mörgelin M. Sommarin Y. WennerGren Int. Ser. 1998; 72: 113-122Google Scholar, 6Underhill C. J. Cell Sci. 1992; 103: 293-298Crossref PubMed Google Scholar, 7Masselis-Smith A. Belch A.R. Mant M.J. Turley E.A. Pilarski L.M. Blood. 1996; 5: 1891-1899Crossref Google Scholar, 8Jackson R.L. Busch S.J. Cardin A.D. Physiol. Rev. 1991; 71: 481-539Crossref PubMed Scopus (952) Google Scholar). Among a variety of HABPs that have been reported to date, SHAPs are the first and so far the only proteins covalently bound to HA (9Yoneda M. Suzuki S. Kimata K. J. Biol. Chem. 1990; 265: 5247-5257Abstract Full Text PDF PubMed Google Scholar, 10Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The SHAP-HA complex was originally discovered from the HA-rich matrix of cultured mouse dermal fibroblasts, and SHAP was found to be derived from the serum supplemented to the culture media (the serum-derived hyaluronan-associated protein) (9Yoneda M. Suzuki S. Kimata K. J. Biol. Chem. 1990; 265: 5247-5257Abstract Full Text PDF PubMed Google Scholar, 11Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar). SHAPs correspond to the heavy chains of plasma inter-α-trypsin inhibitor (ITI) family molecules and are bound to HA via a unique ester bond (10Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 11Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar). The ITI family molecules are synthesized by hepatocytes and secreted into blood at high concentrations (0.15–0.5 mg/ml plasma) (12Mizon C. Balduyck M. Albani D. Michalski C. Burnouf T. Mizon J. J. Immunol. Methods. 1996; 190: 61-70Crossref PubMed Scopus (31) Google Scholar). The heavy chains (HC1, HC2, and HC3) of these molecules are derived from three different genes, and either one or two of them are covalently bound to the light chain, bikunin, to form the ITI family members such as ITI, pre-α-trypsin inhibitor (PαI), and inter-α-trypsin-like inhibitor (13Salier J.P. Trends Biochem. Sci. 1990; 15: 435-439Abstract Full Text PDF PubMed Scopus (119) Google Scholar). Bikunin carries at the serine residue at position 10 an O-glycosidically linked chondroitin sulfate chain, to which the heavy chains are linked via ester bonds of exactly the same type as that in a SHAP-HA complex (10Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Enghild J.J. Salvesen G. Hefta S.A. Thogersen I.B. Rutherfurd S. Pizzo S.V. J. Biol. Chem. 1991; 266: 747-751Abstract Full Text PDF PubMed Google Scholar). During the formation of the SHAP-HA complex, HA is substituted for the chondroitin sulfate chain of bikunin, accompanied by the release of bikunin. Plasma also includes an enzymatic activity catalyzing the transfer of the heavy chains from the chondroitin sulfate to HA (11Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar). Therefore, one can assume that the SHAP-HA complex is formed wherever and whenever plasma encounters HA. The SHAP-HA complex has been found in the HA-rich matrix of expanded cumulus oophorus (15Chen L. Mao S.J.T. McLean L.R. Powers R.W. Larsen W.J. J. Biol. Chem. 1994; 269: 28282-28287Abstract Full Text PDF PubMed Google Scholar) and in the sera and synovial fluids of patients suffering from rheumatoid arthritis (10Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 16Kida D. Yoneda M. Miyaura S. Ishimaru T. Yoshida Y. Ito T. Ishiguro N. Iwata H. Kimata K. J. Rheumatol. 1999; 26: 1230-1238PubMed Google Scholar). Previous studies (15Chen L. Mao S.J.T. McLean L.R. Powers R.W. Larsen W.J. J. Biol. Chem. 1994; 269: 28282-28287Abstract Full Text PDF PubMed Google Scholar, 17Blom A. Pertoft H. Fries E. J. Biol. Chem. 1995; 270: 9698-9701Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) suggested that the ITI family molecules, ITI and PαI, stabilized the HA-rich matrix of cultured cells and cumulus oophorus. Recently, the in vivo implications of the SHAP-HA complex in the extracellular matrix have become evident from our study on bikunin-knockout mice that are unable to form the complex (18Zhuo L. Yoneda M. Zhao M. Yingsung W. Yoshida N. Kitagawa Y. Kawamura K. Suzuki T. Kimata K. J. Biol. Chem. 2001; 276: 7693-7696Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). In the mice, the cumulus oophorus, an investing structure unique to the oocyte of higher mammals, had a defect in forming the extracellular HA-rich matrix, and the ovulated oocyte was unfertilized, leading to severe female infertility (18Zhuo L. Yoneda M. Zhao M. Yingsung W. Yoshida N. Kitagawa Y. Kawamura K. Suzuki T. Kimata K. J. Biol. Chem. 2001; 276: 7693-7696Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). However, it is yet unclear how the SHAP-HA complex contributes to the stabilization of an HA-rich matrix. Details on its molecular structure, particularly the number of SHAP molecules bound to an HA chain, would help us to understand the molecular interactions in the HA-rich matrix, not only between the SHAP-HA complexes but also between the SHAP-HA complex and other matrix components such as TSG-6 (19Mukhopadhyay D. Hascall V.C. Day A.J. Salustri A. Fulop C. Arch. Biochem. Biophys. 2001; 394: 173-181Crossref PubMed Scopus (105) Google Scholar). Here for the first time, we have characterized a structural feature of the SHAP-HA complex isolated from pathological synovial fluid, and we provide evidence for the multiple binding of SHAPs to an HA chain. Materials—Aminocaproic acid, N-ethylmaleimide, phenylmethylsulfonyl fluoride (PMSF), and CsCl were purchased from Nacalai Tesque, Kyoto, Japan. Protease-free Streptomyces hyaluronidase, chondroitinase ABC, the HA-binding protein (HABP) derived from the N-terminal region of bovine aggrecan, the biotinylated HABP, mouse anti-human PG-M/versican monoclonal antibody, 2B1, and mouse anti-human decorin monoclonal antibody, 6B6, were from Seikagaku Corp., Tokyo, Japan. Rabbit anti-human ITI antibody (purified immunoglobulins) was from Dako, Glostrup, Denmark. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (affinity-purified immunoglobulins) was from Jackson ImmunoResearch Laboratories, West Grove, PA. HRP-conjugated streptavidin was from Amersham Biosciences. HRP-conjugated protein A was from Organon Teknika Corp., West Chester, PA. Bovine serum albumin fraction V (protease-free grade) (BSA) was from Miles. Skim milk was from Difco. The 3,3′, 5, 5′-tetramethylbenzidine (TMB) solution and TMB stop solution were from Kirkegaard & Perry Laboratories. Micro-BCA Protein Assay Reagent kit was from Pierce. Western blot chemiluminescence reagent was from PerkinElmer Life Sciences. Hyperfilm ECL film and Sephacryl S-1000 were from Amersham Biosciences. Rabbit anti-human bikunin antibody (purified immunoglobulins) was prepared in this laboratory (18Zhuo L. Yoneda M. Zhao M. Yingsung W. Yoshida N. Kitagawa Y. Kawamura K. Suzuki T. Kimata K. J. Biol. Chem. 2001; 276: 7693-7696Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Purification of the SHAP-HA Complex from Pathological Synovial Fluid—The SHAP-HA complex was purified by a series of CsCl isopycnic centrifugations with increasing initial densities. Synovial fluid (70 ml) from knee joints of three rheumatoid arthritis patients with no record of previous treatment with intraarticular hyaluronan injection, which had been stored immediately after the collection at –80 °C, was mixed with the same volume of extraction buffer containing 0.2 m Tris-HCl (pH 8.0), 8 m guanidine HCl, 10 mm EDTA, 10 mm aminocaproic acid, 10 mm N-ethylmaleimide, and 2 mm PMSF. After stirring overnight at 4 °C, the solution was brought to a density of 1.35 g/ml with solid CsCl. Then a density gradient was established by centrifugation at 40,000 rpm at 10 °C for 48 h (Hitachi 70P-72, rotor RP 70T-403) (the first centrifugation). The gradient was partitioned into 15 fractions (2 ml/fraction) from the bottom, and the fractions were subjected to uronate and protein content analysis by the carbazole reaction (9Yoneda M. Suzuki S. Kimata K. J. Biol. Chem. 1990; 265: 5247-5257Abstract Full Text PDF PubMed Google Scholar) and micro-BCA assay, respectively. The fractions (fractions 1–6 with densities of 1.35–1.46 g/ml) that contained most of the total HA were pooled and subjected to a second centrifugation with an initial density of 1.40 g/ml and subsequent assay for uronate and protein content for each fraction after being partitioned into 15 fractions (2 ml/fraction) from the bottom. Fractions 3–9 that had the high concentrations of HA were pooled and subjected to a third centrifugation with an initial density of 1.42 g/ml. The third gradient was partitioned into 27 fractions (1 ml/fraction), and the contents of HA, total glycosaminoglycan, and protein were measured by periodate/thiobarbituric acid reaction (20Yoneda M. Yamagata M. Suzuki S. Kimata K. J. Cell Sci. 1988; 90: 265-273Crossref PubMed Google Scholar), carbazole reaction, and micro-BCA assay, respectively. SDS-PAGE and Immunostaining—The amount of SHAP in each fraction was determined by Coomassie Brilliant Blue (CB) staining and by immunostaining with anti-ITI antibody. The fraction samples containing 20 or 3 μg of HA for CB staining and immunostaining, respectively, were mixed with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0 °C. After centrifugation at 10,000 rpm, the pellet was dissolved in 50 μl of 0.1 m sodium acetate buffer (pH 6.0) containing 0.5 turbidity reducing units of protease-free Streptomyces hyaluronidase, then incubated at 60 °C for 2 h or dissolved in 50 μl of 0.1 m sodium acetate buffer (pH 7.0) containing 50 milliunits of chondroitinase ABC, and then incubated at 37 °C for 2 h. The samples were concentrated in vacuo and mixed with the sample buffer for SDS-PAGE (1% SDS, 50 mm Tris-HCl (pH 6.8), 0.1% dithiothreitol, and 20% glycerol), before being incubated at 37 °C for 1 h. Electrophoresis was carried out in a 7.5% gel under reducing conditions. After electrophoresis, the proteins on the gel were stained with CB or transferred to a Hybond extra C membrane for immunostaining. The membrane was blocked with 10% skim milk, 0.1% Tween 20 in PBS (PBS-T) at 40 °C for 2 h, and then incubated with rabbit anti-human ITI antibody (1:2000 in PBS-T) at room temperature for 1 h. After several washes with PBS-T, the membrane was further incubated with HRP-conjugated protein A (1:4000 in PBS-T). The immune complexes were visualized by enhanced chemiluminescence assay and exposed to Hyperfilm ECL. Quantification of SHAP in the SHAP-HA Complex—Nunc Maxisorp microtiter plates were coated with HABP (2 μg/ml in 0.1 m sodium carbonate buffer (pH 8.25)) at 4 °C for 15 h. The wells were washed twice with 200 μl of PBS-T, followed by blocking with 200 μl of 3% BSA in PBS-T at room temperature for 2 h. After three washes with 200 μl of PBS-T, 50 μl each of sample (500 ng HA/ml) in 1% BSA/PBS-T was added to the well, and the plates were incubated at 37 °C for 2 h. After a wash, 25 μl of rabbit anti-human ITI antibody (diluted 1:2000 with 1% BSA/PBS-T) and 25 μl of HRP-conjugated goat anti-rabbit immunoglobulins antibody (diluted 1:1500 with 1% BSA/PBS-T) were added to each well and incubated at 37 °C for 1 h. The wells were washed 3 times and then incubated with 50 μl each of TMB solution at 37 °C for 20 min. The reaction was stopped by adding 50 μl of 1 m HCl, and the absorbance at 450/620 nm (A at 450 nm minus A at 620 nm) was measured on an immunoMini NI-2300 spectrophotometer. The assay was performed in triplicate. Quantification of HA in the SHAP-HA Complex—Nunc Maxisorp microtiter plates were coated with 50 μl each of rabbit anti-human ITI antibody (22 μg/ml in 20 mm NaHCO3 (pH 9.5)) at 4 °C for 15 h and then blocked with 1% BSA in PBS-T for 60 min at 37 °C. After three washes with PBS-T, 50 μl each of sample (300 ng HA/ml) in 1% BSA/PBS-T was applied to each well and incubated at 37 °C for 60 min. The wells were washed and incubated with 50 μl each of biotinylated-HABP (diluted 1:2000 with 1% BSA/PBS-T) at 37 °C for 60 min. After a wash with PBS-T, 50 μl of HRP-streptavidin (1:500) was added to each well, and the plates were further incubated at 37 °C for 60 min. Color development was achieved by incubating with 50 μl of TMB solution at 37 °C for 5 min and then stopped with 50 μl of 1 m HCl. The absorbance at 450/620 nm (A at 450 nm minus A at 620 nm) was measured. The assay was performed in triplicate. Immunoprecipitation of the SHAP-HA Complex—Anti-ITI antibody (110 μg) in 1.5 ml of PBS (pH 7.4) was mixed with an equal volume of PBS-equilibrated cellulofine-protein A beads and incubated at room temperature for 2 h with gentle mixing. The beads were collected by centrifugation followed by five washes with 3 volumes each of PBS. The samples (1.5 ml) including 7.5 μg of HA in PBS with 2 mm PMSF were mixed with the anti-ITI antibody-coated beads and incubated overnight at 4 °C with gentle mixing. The beads were collected by centrifugation and washed 5 times with 3 volumes each of PBS. The supernatant and the first washing solution were pooled for the measurement of unbound free HA. The SHAP-HA complex bound on the beads was treated with 50 mm NaOH with 2 mm PMSF at room temperature for 4 h to release HA (10Zhao M. Yoneda M. Ohashi Y. Kurono S. Iwata H. Ohnuki Y. Kimata K. J. Biol. Chem. 1995; 270: 26657-26663Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The beads were washed once with 50 mm NaOH, and the two NaOH solutions were pooled. The amounts of unbound HA (free HA) and bound HA (in the form of the SHAP-HA complex) were measured by the carbazole reaction. After concentration in vacuo, HA was precipitated with ethanol, dried briefly, dissolved in 30 μl of TAE (40 mm Tris acetate, 1 mm EDTA) buffer, and then subjected to 0.5% agarose gel electrophoresis as described below. Agarose Electrophoresis and Western Blotting of the SHAP-HA Complex and HA—The SHAP-HA complex preparation (containing 30 μg of HA) was precipitated with ethanol, briefly dried, and then dissolved in 30 μl of TAE. If necessary, the precipitate of the preparation was dissolved in 30 μlof0.2 m NaOH and incubated at room temperature for 4 h to completely break down the ester bond between SHAP and HA. The released HA was precipitated again with ethanol, dissolved in 30 μl of TAE buffer, and then subjected to 0.5% agarose gel electrophoresis (21Lee H.G. Cowman M.K. Anal. Biochem. 1994; 219: 278-287Crossref PubMed Scopus (257) Google Scholar). After electrophoresis, the gel was incubated in a freshly prepared solution (176 mg of l-ascorbic acid and 13.9 mg of FeSO4 in 500 ml of TAE buffer) for 30 min to degrade the HA into low molecular weight HA. After two washes with TAE buffer for 20 min, the HA in the gel was blotted onto a Hybond N+ membrane by capillary action. HA was directly visualized by staining with 0.5% Alcian blue in 2% acetic acid for 15 min, followed by destaining with 2% acetic acid. Otherwise, the membrane was blocked with 20% skim milk in PBS-T overnight at room temperature with gentle shaking and incubated with biotinylated HABP (1 μg/ml) in PBS with 1% BSA at room temperature for 2 h and then with HPR-streptavidin (1:1000) in PBS with 1% BSA at room temperature for 1 h. The HA was finally visualized by enhanced chemiluminescence assay and exposed to Hyperfilm ECL. Gel Filtration of the SHAP-HA Complexes—The SHAP-HA complex preparation was dialyzed against distilled water. An aliquot of the dialyzed preparation was treated with 0.2 m NaOH for 2 h at room temperature, followed by neutralization with HCl and subsequent addition of 10× PBS buffer to give the conventional PBS solution. For control, all additives were mixed first, and finally the aliquot was added. The sample was applied on to an analytical Sephacryl S-1000 column (0.7 × 30 cm). The column was eluted with the conventional PBS solution at a flow rate of 12–15 ml/h, and 0.5-ml fractions were collected. 4 and 10 μl of each fraction were diluted with the PBS solution to make a total volume of 50 μl and then subjected to quantification of HA and SHAP, respectively. Quantification of HA by Inhibitory ELISA—The inhibitory ELISA kit for HA was provided by Seikagaku Corp., Tokyo, Japan. Briefly, the microtiter plates were coated with HA-conjugated BSA and then blocked with BSA. To each well, the samples or various concentrations of HA (HA standards) were added, together with a biotinylated HABP solution. After incubation, the plate was washed and further incubated with peroxidase-conjugated streptavidin. Finally, a color was developed, and the absorbance was measured as described above. Electron Microscopic Observation of the SHAP-HA Complex—Negative staining of the SHAP-hyaluronan complex for electron micrographs and evaluation of the data were carried out as described previously (22Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (136) Google Scholar). For negative staining, sample preparations (typical concentrations of about 10 μg/ml in Tris-buffered saline) were adsorbed to 400-mesh carbon-coated copper grids, washed briefly with water, and stained on two drops of freshly prepared 0.75% uranyl formate. The grids were rendered hydrophilic by glow discharge at low pressure in air. Specimens were observed in a Jeol JEM 1230 electron microscope operated at an 80-kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 CCD camera. Molecular masses of globular protein domains from negatively stained images were estimated as described previously (22Engel J. Furthmayr H. Methods Enzymol. 1987; 145: 3-78Crossref PubMed Scopus (136) Google Scholar). Purification of the SHAP-HA Complex from Pathological Synovial Fluid—The synovial fluid from rheumatoid arthritis patients was highly viscous and contained a variety of proteins at high concentrations. The SHAP-HA complex was isolated under dissociative conditions with 4 m guanidine HCl by three sequential CsCl isopycnic centrifugations with initial densities of 1.35, 1.40, and 1.42 g/ml, respectively (Fig. 1A). After each centrifugation and subsequent fractionation, the density and uronate, HA, and protein contents of each fraction were measured. Fractions were numbered from the bottom to the top. After the first centrifugation, most of the proteins were recovered in the lower density fractions (ρ <1.35 g/ml) (Fig. 1A). The higher density fractions (fractions 1–6) that contained less protein and more uronate were pooled and subjected to a second centrifugation. After the second centrifugation, we noticed that the relative content of protein to uronate in each fraction was not constant but tended to increase along with the decline in density (Fig. 1B). The fractions 3–9 of the second gradient were pooled and further purified by a third centrifugation. The fractions 5–15 of the third gradient that had densities between 1.5 and 1.37 g/ml contained most of the total HA, but the protein-to-HA ratio still showed an increasing tendency, from 0.084 for fraction 5 to 0.148 for fraction 15 (Fig. 1C). Two-thirds each of the fractions 5–15 were pooled as the purified SHAP-HA complex. Comparing uronate and protein contents of the pooled fractions among the three centrifugations, we found that the relative amounts of protein to uronate decreased significantly between the first and second centrifugations but only a little between the second and third centrifugations (Table I). There was no further decrease when we performed the fourth centrifugation and compared the values with those for the third centrifugation (data not shown). Judging from these values and the distribution patterns of protein, uronate, and HA after each centrifugation, we concluded that the SHAP-HA complex in the starting synovial fluid of rheumatoid arthritis patients was mostly recovered in fractions 5–15 of the third gradient with the least contamination by other components. In this experiment, the final SHAP-HA complex preparation with 70 ml of synovial fluid (obtained from three rheumatoid arthritis patients) as starting material contained 3.08 mg of protein and 56 mg of HA.Table IThe protein, uronate, and HA contents of the pooled fractions after the three CsCl isopycnic centrifugationsCentrifugationProteinUronateHAProtein/uronateProtein/HAmg/mlmg/mlmg/ml10.6161.7120.36020.1971.1720.16830.1400.6951.2960.2010.108 Open table in a new tab Characterization of the Isolated SHAP-HA Complex—The purity of the SHAP-HA complex preparations was examined by CB staining and immunostaining after SDS-PAGE under reducing conditions. The pooled fraction after the second centrifugation without Streptomyces hyaluronidase digestion only gave the protein bands migrating faster than the SHAP bands (Fig. 2A, lane 1). The appearance of those bands such as the major one having molecular mass of 50 kDa was not altered by Streptomyces hyaluronidase digestion, and therefore, those protein bands were likely contaminants. The Streptomyces hyaluronidase digestion of the fraction gave two new bands having molecular masses of 75 and 85 kDa, which were stained with CB strongly and weakly, respectively (Fig. 2A, lane 2). They appeared to correspond to the SHAPs derived from HC1 and HC2 of ITI, respectively, according to the sensitivity to hyaluronidase digestion and the reported molecular sizes for human HC1 and HC2 (12Mizon C. Balduyck M. Albani D. Michalski C. Burnouf T. Mizon J. J. Immunol. Methods. 1996; 190: 61-70Crossref PubMed Scopus (31) Google Scholar, 23Rouet P. Daveau M. Salier J.P. Biol. Chem. Hoppe-Seyler. 1992; 373: 1019-1024Crossref PubMed Scopus (27) Google Scholar). The fractions 5–14 on the third gradient, the representative fractions, did not give the protein bands migrating faster than the SHAP bands with or without the hyaluronidase digestion (Fig. 2B), which suggested that the third centrifugation was essential to purify the SHAP-HA complex from those contaminants. After Streptomyces hyaluronidase digestion, the fractions only gave the two bands having molecular masses of 75 and 85 kDa upon CB staining (Fig. 2B, lanes 1 and 3). Both of them were stained with the anti-human ITI antibody, confirming that they corresponded to ITI HCs (Fig. 2C). The results are consistent with our previous study showing that the SHAP-HA complex in human pathological synovial fluids was composed of both the HC1 and HC2 of the ITI (11Huang L. Yoneda M. Kimata K. J. Biol. Chem. 1993; 268: 26725-26730Abstract Full Text PDF PubMed Google Scholar). The CB staining pattern indicated that the synovial SHAP-HA complex involves more HC1 than HC2. Chondroitinase ABC digestion (Fig. 2B, lanes 2 and 4) did not yield any further bands except for the own enzyme bands (Fig. 2B, lane 6), which almost but not completely eliminated the possibility of contamination with chondroitin sulfate proteoglycans (Fig. 2B, lanes 2 and 4). This was also confirmed by no immunoreactivity on the membranes of chondroitinase ABC digested fraction 5 and 14 samples with antibodies against human PG-M/versican, human decorin, and human bikunin (data not shown). The Differences in the Density of the SHAP-HA Complex Fractions Are Due t
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