3Course of Advanced Biosciences, Graduate School of Humanities and Sciences, 4Department of Biochemistry, St. Marianna University School of Medicine, Sugao, Miayamae-ku, Kawasaki, Kanagawa, Japan, 5Department of Nutrition and Food Science and 6Department of Chemistry, Faculty of Sciences, Ochanomizu University, Otsuka, Bunkyo-ku, Tokyo 1128610, Japan
Received on January 5, 2000; revised on February 28, 2000; accepted on March 6, 2000.
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Abstract |
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Key words: glycosylation/liver regeneration/tissue remodeling/collagen binding/vitronectin
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Introduction |
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Vitronectin (VN) is a multi-functional adhesive glycoprotein, that originates mainly in hepatocytes and circulates in the blood stream at high concentrations (0.2 mg/ml in humans). VN regulates the blood systems related to protease cascades such as cell lysis by complement, coagulation and fibrinolysis (reviewed in Tomasini and Mosher, 1990; Preissner, 1991
). VN is also found in the extracellular matrix of most tissues and is considered to play a role in cell adhesion, cellular motility and matrix remodeling. Tissue VN is considered to be present as an active multimeric form, and interactions with various matrix ligands, such as various types of integrins on the cell surfaces, type 1 plasminogen activator inhibitor, and urokinase receptor to regulate pericellular proteolysis (reviewed in McKeown-Longo and Panetti, 1996
; Seiffert, 1997
; Preissner and Seiffert, 1998
), are responsible for these functions. VN can also bind to various types of collagen through its conformational transition from the native inactive form to an active form (Gebb et al., 1986
; Izumi et al., 1988
; Ishikawa-Sakurai and Hayashi, 1993
). In our preceding study, purified human plasma VN and its recombinant domains, hemopexins II and I, bound to type I collagen under physiological conditions (Yoneda et al., 1998
). The collagen binding activity was affected by the presence or absence of N-glycan covalently linked to VN. The biological significance of the interaction with collagen is not yet clear.
In this study, we chose the liver regeneration induced by two-thirds partial hepatectomy as a model system to study whether and how VN plays a role in tissue remodeling after hepatectomy and if glycosylation is involved in the physiological processes. In the early stage of liver regeneration, the synthesis of total DNA increased while the synthesis of total glycoproteins decreased within 48 h after partial hepatectomy (Okamoto and Akamatsu, 1977). The contradictory decrease of total glycoprotein synthesis in regenerating rat liver is due to the attenuation of the oligosaccharide transferase activity in microsomes (Oda-Tamai et al., 1985
). Alterations in the glycan structure of total hepatic glycoproteins have been also suggested during liver regeneration (Kato and Akamatsu, 1984
, 1985; Ishii et al., 1985
). However, the changes in the glycans of a particular glycoprotein have not been well characterized. For these reasons, we investigated the changes of rat plasma VN after partial hepatectomy with particular focus on the relationship between the glycosylation and collagen binding activity of VN. We found evidence that the glycosylation of VN changed in response to the surgery, and that it dramatically enhanced collagen binding, which may contribute to the matrix incorporation of VN and subsequent repair or remodeling processes.
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Results |
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Amino acid composition
To compare the polypeptide portion, amino acid compositions of rat VNs were analyzed and are summarized in Table I. The similarity in polypeptides of the three VNs was compared by the index of composition divergence (Black and Harkins, 1977). The composition divergence taking VN of nonoperated rat as 0.0 was 0.049 and 0.053 for VNs from sham-operated and partially hepatectomized rats, respectively. When taking VN of sham-operated rat as 0.0, the composition divergence of VN from partially hepatectomized rat was 0.040. All the values were smaller than 0.06, and it was predicted that the three VNs would have high homology among the primary sequences. These VNs showed the same N-terminal 6 amino acid sequences identical to that predicted from cDNA (Otter et al., 1995
), DQESXK, where X is predicted to be cysteine according to the cDNA sequence.
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The pI of VN from nonoperated rat was pH 4, but that of VN from partially hepatectomized rat was pH 6. VN from sham-operated rat was slightly above pH 4.
Interaction of VNs with various HRP-lectins
As is summarized in Table III, all VNs were reactive with N-glycan-specific lectins, Con A, LCA, and RCA, indicating that these VNs contain N-linked complex-type oligosaccharides, whose innermost N-acetyl-D-glucosamine residue bound to asparagine is possibly fucosylated like porcine VN (Yoneda et al., 1993) by the reactivity with LCA and the presence of L-fucose in these VNs (Table III). Reactivity toward L-PHA, E-PHA, and PVL varied remarkably among VNs, and the result of densitometry is shown in Figure 2. As shown in Figure 2A, VN of partially hepatectomized rats showed marked reactivity with L-PHA, but VNs of sham-operated and nonoperated rats reacted only slightly, suggesting that tri- or tetraantennary lactosamine structures drastically multiplied after partial hepatectomy. VN of partially hepatectomized rats showed reactivities with E-PHA two times greater than those of sham-operated and nonoperated rats (Figure 2B), suggesting that the biantennary or triantennary lactosamine structures with bisecting GlcNAc increased after partial hepatectomy. These results together indicate that the branching of N-linked oligosaccharides of VN increased after partial hepatectomy by attachment of multiple GlcNAc side chains, which is consistent with the increased GlcNAc concentration in the carbohydrate composition (Table III). On the other hand, VN of partially hepatectomized rats showed considerably weaker reactivities with PVL than sham-operated VNs did (Figure 2C). PVL was found to recognize nonreducing sialyl residues besides nonreducing GlcNAc, depending on the number rather than the linkage of sialic acids (Ueda et al., 1999
). Positive staining with PVL was lost after desialylation of VNs (Table III), indicating that the different staining intensities of these VNs with PVL suggest the extent of differential sialylation of these VNs, which agreed with the decreased NeuAc concentrations in postoperative VNs (Table II).
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Binding activity of purified VNs to type I collagen
VNs used in this study were urea-unfolded and then refolded under high salt concentration, which is considered to produce a monomeric form that is active in ligand binding (Zhuang et al., 1996a,b). The binding of purified VNs to type I collagen was measured by ELISA using type I collagen-coated plates.
The raw absorbance data of collagen-bound VN was corrected for the relative antibody reactivity of each VN shown in Figure 3A, and the corrected absorbances of bound VN are presented in Figure 3B. VN from partially hepatectomized rats was found to exhibit much greater binding to collagen than VN from sham-operated rats and nonoperated rats (Figure 3B). As shown in Figure 3C, VNs from partially hepatectomized and sham-operated rats showed about 3 and 1.6 times higher binding, respectively, to collagen than that of VN from nonoperated rats. The reactivity of VN from partially hepatectomized rats was about 2 times that of sham-operated rats.
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Discussion |
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Plasma VN obtained from partially hepatectomized rats was found to have low carbohydrate concentrations, which suggests it is less glycosylated. The decreased N-glycosylation of VN at 24 h after partial hepatectomy could be attributed to the attenuation of the oligosaccharide transferase activity in microsomes (Oda-Tamai et al., 1985), which also causes the decrease of total glycoprotein synthesis in regenerating liver (Okamoto and Akamatsu, 1977
). In the early stage after partial hepatectomy, activities of several glycosyltransferases have been reported to increase, i.e., N-acetylglucosaminyltransferases III, V (Miyoshi et al., 1995
), and I (Okamoto and Akamatsu, 1978
), galactosyltransferase (Bauer et al., 1976
) and sialyltransferase (Ip, 1979
; Serafini-Cessi, 1977
). In this study, molar ratios of GlcNAc to total carbohydrates of VNs markedly increased (Table II, in parentheses), and the reactivities with L-PHA and E-PHA were enhanced after partial hepatectomy (Figure 2). The results suggest an increased branching of oligosaccharides of VN synthesized by partially hepatectomized rat liver, whereas the number of oligosaccharides transferred to the peptide decreased. The changes of VN glycosylation observed after partial hepatectomy agree with the reported changes of related enzyme activities, and are considered to be caused by the sum of physiological changes of various glycosyltransferases involved in the oligosaccharide transfer and processing during liver regeneration.
Two of three potential N-glycosylation sites in human VN are conserved among mammalian VNs, one of which is located in the hemopexin I domain and another in a connecting region (Yoneda et al., 1996). In our previous study, the collagen binding domains of VN were identified as hemopexin II and I domains (Yoneda et al., 1998
). It is unclear whether the binding sites of both hemopexin II and I domains are affected by glycosylation, but the enhancement of the hemopexin I site at least might increase the avidity of the two sites to generate greater binding to collagen.
A similar modulation of collagen binding activity was suggested for osteonectin (Kelm and Mann, 1991), an extracellular matrix-associated, counter-adhesive glycoprotein expressed in adult tissues undergoing remodeling and renewal, and during embryonic development. Bone osteonectin possesses a high-mannose type glycan and binds to types I, III, and V collagen, whereas platelet osteonectin, which possesses a sialylated complex-type glycan, does not bind to any type collagen (Kelm and Mann, 1991
). The sialooligosaccharides of VN and osteonectin may modulate the interaction with collagen of these adhesive and counter-adhesive glycoproteins due to the anionic charges of sialic acids. The isoelectric points of the three VNs differed considerably, and the pI of sham-operated rat VN changed from 4 to 6 after neuraminidase treatment (data not shown), which was close to the pI of VN from partially hepatectomized rats. It is therefore suggested that sialic acids significantly affect the net charge of the VN molecule and may electrostatically interfere with the collagen binding of VN. The involvement of the other glycan moieties and the interdomain interaction in the modulation of collagen binding remains to be clarified.
It is well documented that the liver is the major site of biosynthesis of plasma glycoproteins (Macbeth et al., 1965; Bekesi and Winzler, 1967
) and that partial hepatectomy causes a transient decrease in the total amount of plasma proteins (Bresnick, 1971
). The ratio of purified VN to total plasma proteins (w/w, %) decreased in the early stage of liver regeneration (Figure 1C), suggesting that the decrease in amount of the VN should not be ascribed simply to the decreased liver mass. Since the mRNA level as well as the protein level of vitronectin were reported to be unchanged in liver remnants after partial hepatectomy until 24 h (Kim et al., 1997
), the decrease could result from enhanced removal of VN from blood circulation by asialoglycoprotein receptors or by matrix incorporation. Alternatively, the decreased ratio may be caused by reduction in the efficiency of purification, though heparin binding activity was not affected by de N-glycosylation of VN in our previous observation (Yoneda et al., 1998
).
Recent evidence indicates that VN may be involved in wound healing or tissue remodeling processes associated with injury or inflammation (Inuzuka et al., 1994; Seiffert, 1997
; Preissner and Seiffert, 1998
). One example is that VN expression in murine and human liver is upregulated in acute and chronic inflammation, suggesting that VN is regulated as an acute phase reactant (Seiffert et al., 1995
, 1996; Seiffert, 1997
). Changes of VN in plasma in various liver disorders and inflammation have also been reported. Plasma VN levels decline in chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma with cirrhosis (Kemkes-Matthes et al., 1987
; Inuzuka et al., 1992
; Kobayashi et al., 1994
), while the collagen-binding VN in plasma increases in these diseases, compared with those of healthy controls (Inuzuka et al., 1992
; Yamada et al., 1996
), and the amount of collagen-binding VN was significantly correlated with certain fibrous markers (Yamada et al., 1996
). The co-deposition of VN with collagen fibrils has been observed in hepatic fibrosis (Kobayashi et al., 1994
) and cirrhosis (Inuzuka et al., 1992
), suggesting that the collagen binding of VN is significantly changed during these pathological processes. These observations suggest the roles of VN may lie in controlling the extracellular reactions during remodeling of damaged tissue, or alternatively, VN may causally relate to the progression of these diseases. In this context, the collagen binding activity may participate in the incorporation of VN into the extracellular matrix, which may function as the initial step of VN entry into the tissue and/or accelerating hepatic fibrogenesis (Inuzuka et al., 1994
). Depending on glycosylation diversities occurring in nature, the collagen binding of VN can be exhibited diversely by physiological processes. This study proposes the possibility that modulation of biological activity of VN is performed through altered glycosylation in the tissue remodeling processes during liver regeneration, and possibly, in various pathophysiological states. Such a glycomodulation may cooperate with or compensate for the regulated expression of VN.
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Materials and methods |
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Animals
Male Wistar rats aged 56 weeks (weighing about 120 g; Nihon Clea, Tokyo, Japan or Nihon Rat, Tokyo, Japan) were maintained in a room at a constant temperature (23.5°C) with 12 h each light (6:0018:00) and darkness. Two-thirds partial hepatectomy was performed under diethyl ether anesthesia as reported previously (Oda-Tamai et al., 1985). Sham-operated animals were anesthetized, and their livers were exposed completely outside the peritoneum and manipulated, but not removed. Plasma was collected from partially hepatectomized and sham-operated rats at the indicated times after the operation. Plasma samples were stored at 80°C until use.
Purification of VN from rat plasma
Rat VN was purified from plasma of partially hepatectomized, sham-operated, or nonoperated rats as described previously (Yatohgo et al., 1988). VN was eluted from a heparin column in a urea-denatured condition and was then refolded in the presence of 2 M NaCl to obtain a monomer form of VN that is active in ligand binding (Zhuang et al., 1996a
).
Protein determinations
The amounts of purified VN were estimated by means of absorbance measurements at 280 nm with 1 cm path-length cells, using the absorption coefficient of 1.38 at 1 mg/ml (Dahlback and Podack, 1985). The amount of total protein in plasma was determined as described previously (Oda-Tamai et al., 1985
).
SDSPAGE and isoelectric focusing
SDSPAGE was carried out using 9% polyacrylamide gel according to the method of Laemmli (Laemmli, 1970) under reducing conditions. Isoelectric focusing was performed according to the manufacturers instruction.
Amino acid analysis
Purified plasma VN (7 µg) was dot-blotted onto PVDF membrane. The band was stained with Coomassie brilliant blue and excised from the membrane. The membrane was destained and extensively washed with milli-Q water. Hydrolysis was carried out in vacuo with a vapor of 6 M HCl at 110°C for 24 h. After hydrolysis, amino acids were derivatized with 4-[4-(dimethylamino)phenylazo]benzenesulfonyl-chloride (DABS-Cl). The DABS-derivatized amino acids were analyzed with reverse-phase HPLC using a CCD-ODS column (4.0 x 125 mm, Ciba-Corning) after evaporation according to manufacturers instructions (Jusco Co., Tokyo, Japan). The index of composition divergence (D) was calculated according to Black and Harkins (Black and Harkins, 1977) using the equation, D = [
(xiA xiB)2]1/2, where xiA and xiB mean mol% of amino acid "i" in proteins A and B, respectively.
Carbohydrate analysis
VNs (10 µg) were placed in a glass tube that had been washed with boiling 50% nitric acid and water and dried in vacuo. For neutral sugar and amino sugar analysis, hydrolysis was carried out in vacuo with a vapor of 2 M HCl, 2 M trifluoroacetic acid for 4 h at 100°C. After hydrolysis, the mixture was N-acetylated and then reacted with a fluorescent probe, 2-aminopyridine, and carbohydrate was analyzed as previously reported (Suzuki et al., 1990). Sialic acid component analysis was performed according to the method of Hara et al. (1987)
after hydrolysis of the sample (25 µg) with 0.025 M HCl at 80°C for 1 h.
Interactions of VNs with HRP-lectins
VNs were dot-blotted onto a PVDF membrane in amounts of 0.10.2 µg/100 µl and reacted with HRP-lectins as previously described (Kitagaki-Ogawa et al., 1990). The bound HRP-lectins were developed with 4-chloro-1-naphthol/H2O2. The staining intensities were measured by a refractive densitometer, Shimadzu CS9300PC (Shimadzu Seisakusho, Kyoto, Japan), at an absorbance of 370 nm for lectin staining, or at 550 nm for CBB staining.
Assays for binding of VN to immobilized type I collagen
Collagen binding activities were assayed by ELISA essentially according to the method reported previously (Gebb et al., 1986; Yoneda et al., 1998
). The wells of microtiter plates (Immulon 1, from Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 µl aliquots of a solution of type I collagen (10 µg/ml in 0.1 M carbonate buffer, pH 9.5) for 3 h at room temperature. The wells were blocked with 5% BSA in 10 mM TrisHCl buffer (pH 7.5) containing 140 mM NaCl (TBS) overnight at 4°C, and various concentrations of purified VN or enzymatically deglycosylated VN (50 µl) were added to each well, followed by incubation for 1 h. After washing with TBS three times, we measured the amount of rat VN bound to immobilized collagen with an HRP-conjugated rabbit anti-human VN IgG and ELISA. In some cases, a combination of unconjugated rabbit anti-human VN IgG and HRP-conjugated anti-rabbit IgG (sheep) was used instead of HRP-anti-human VN IgG. As a control, ELISA was carried out using buffer solution devoid of VN. The antibody reactivity of each VN was measured by directly immobilizing various concentrations of VN (0.00180.13 µg/50 µl) to the well and carrying out ELISA, which was used for the correction of antibody reactivity of bound VN in collagen-ELISA.
Calculation method
When the reactivity of each VN to rabbit anti-human VN IgG was measured, the antibody reactivity of these VNs differed from each other, although immobilization efficiency of these VNs to the well had not differed in the concentration range examined as detected by 125I-labeled each VN (our unpublished observations). The absorbance of collagen-bound VN was thus corrected for the antibody reactivity of each VN. The relative antibody reactivity was calculated from the absorbance ratio of directly immobilized VN from nonoperated rats to that from partially hepatectomized rats, or sham-operated rats, at the same VN concentration. For enzymatically deglycosylated VN, the relative antibody reactivity was calculated from the absorbance ratio of control VN to enzymatically deglycosylated VN. The absorbance of collagen-bound VNs was corrected by multiplying the relative antibody reactivity, and the corrected A490 absorbance was plotted versus VN concentration.
Glycosidase digestion of rat VN
For desialylation, nonoperated rat VN (0.8 mg) was dialyzed against 0.05 M acetate buffer containing 4 mM CaCl2 (pH 5.0) and 0.6% (w/w) 2-mercaptoethanol and digested with neuraminidase (0.02 unit) at 37°C for 24 h. To remove N-linked oligosaccharides, nonoperated rat VN (0.3 mg) in 20 mM phosphate buffer (pH 7.7) containing 130 mM NaCl, 5 mM EDTA and 0.6% (w/w) 2-mercaptoethanol was digested with N-glycosidase F (2 U) at 37°C for 48 h. The digestions were performed without each enzyme for control. Desialylation and deN-glycosylation were ascertained by both the behavior on SDSPAGE and the loss of reactivity with biotin-PVL or HRP-Con A, respectively, on Western blotting.
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Acknowledgments |
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Abbreviations |
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Footnotes |
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2 To whom correspondence should be addressed
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References |
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