The changes in glycosylation after partial hepatectomy enhance collagen binding of vitronectin in plasma

Haruhi Uchibori-Iwaki3, Atsuko Yoneda1,3, Sachie Oda-Tamai4, Shigemi Kato4, Nobu Akamatsu4, Megumi Otsuka5, Kotono Murase6, Kyoko Kojima6, Risa Suzuki6, Yuko Maeya3, Mayumi Tanabe3 and Haruko Ogawa2,3

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 112–8610, Japan

Received on January 5, 2000; revised on February 28, 2000; accepted on March 6, 2000.


    Abstract
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Vitronectin is a multifunctional glycoprotein present in the extracellular matrix and plasma. Changes in rat vitronectin were studied during liver regeneration after partial hepatectomy. Carbohydrate concentrations of vitronectin decreased to 2/3 of sham-operated rats at 24 h after partial hepatectomy. Carbohydrate composition and lectin reactivity indicated that N-glycosylation and sialylation of vitronectin changed markedly after partial hepatectomy, while amino acid composition did not change significantly. We previously showed that deN-glycosylation of vitronectin in vitro affects collagen binding among various ligands (Yoneda et al., Biochemistry (1998) 37, 6351–6360). Vitronectins from partially hepatectomized rats at 24 h were found to exhibit markedly enhanced binding to type I collagen. The effect of sialylation on collagen binding was further examined using enzymatically deglycosylated vitronectin of nonoperated rats. Collagen binding increased by 1.2 times after deN-glycosylation of vitronectin, while it increased more than 2.9 times after desialylation. Various glycosyltransferases in liver are known to change after partial hepatectomy, including the attenuation of N-oligosaccharide transferase. The findings therefore suggest that the collagen binding of vitronectin is modulated by the alteration of peptide glycosylation caused by postoperative physiological changes of glycosyltransferases and that the change may contribute to tissue remodeling processes.

Key words: glycosylation/liver regeneration/tissue remodeling/collagen binding/vitronectin


    Introduction
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Tissue homeostasis has been considered to depend on spatially and temporally controlled expression of multifunctional adhesive glycoproteins and their receptors. Many studies have been performed to follow the changes of expression of matrix molecules during tissue regeneration, inflammation and disease (Kato et al., 1992Go; Hughes, 1997Go; Seiffert, 1997Go). On the other hand, there is increasing evidence that glycosylations post-translationally modulate biological phenomena by altering the binding activity, the binding specificity, or controlling the stability of various glycoproteins through biosignaling functions of oligosaccharides (Blithe, 1993Go; Varki, 1993Go). However, our knowledge of the structure–function relationship of vitronectin is presently limited to the peptide portion.

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, 1990Go; Preissner, 1991Go). 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, 1996Go; Seiffert, 1997Go; Preissner and Seiffert, 1998Go), 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., 1986Go; Izumi et al., 1988Go; Ishikawa-Sakurai and Hayashi, 1993Go). 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., 1998Go). 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, 1977Go). 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., 1985Go). Alterations in the glycan structure of total hepatic glycoproteins have been also suggested during liver regeneration (Kato and Akamatsu, 1984Go, 1985; Ishii et al., 1985Go). 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.


    Results
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Purification of plasma VN from nonoperated, sham-operated, and partially hepatectomized rats
Each purified VN showed one band on SDS–PAGE, as shown in Figure 1A. Plasma VN 24 h after partial hepatectomy (lane c) shifted to a low migration position compared to VNs purified from plasma of nonoperated (lanes a and g), partially hepatectomized rats (lanes b–f) and sham-operated rats (lane h) collected at different times. The migration positions of VN suggest that the molecular mass of VN had shrunk to 65 kDa at 24 h after partial hepatectomy from 68–69 kDa for other VNs, as judged from the migration position of molecular weight markers, although the estimated molecular weights may not necessarily reflect the correct molecular size of VN because of the unusual electrophoretic behavior of its peptide (Nakashima et al., 1992Go).



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Fig. 1. Purification of VNs from plasma of nonoperated, sham-operated, and partially hepatectomized rats. (A) SDS–PAGE of purified VNs. VN from nonoperated rats (a) and (g); VN from partially hepatectomized rats at 6 h (b), 24 h (c), 72 h (d), 144 h (e), 240 h (f) after operation, VN from sham-operated rat at 24 h after operation (h). Molecular weight markers (in Da) are shown on the left and right. (B) yields of purified VN (mg) per ml of plasma at indicated times. (C) ratios of purified VN to total plasma proteins (w/w, %) at indicated times. Symbols used in (B) and (C): dotted bar, VN from nonoperated rat plasma; open bar, VN from sham-operated rat plasma; solid bar, VN from partially hepatectomized rat plasma. Purification was performed from pooled plasma of 2–3 animals at each point. Yields of VN and total plasma protein were measured by protein determinations as described under Materials and methods.

 
Yields of purified VN from post-operative plasma collected at different time intervals are shown in Figure 1B. At 24 h after operation, the VN yield of partially hepatectomized rats had decreased to 1/3 that of sham-operated rats, and it was restored by 240 h when regeneration of the liver was completed. In sham-operated rats, the yield of VN was maximum at 6 h and then gradually decreased. The percentages of purified VN in total plasma protein (w/w, %) changed as shown in Figure 1C. During 6–240 h, the total protein concentration in plasma was within a 41–50 mg/ml range for sham-operated rats while it increased from 27 mg/ml at 24 h to 47 mg/ml at 240 h for partially hepatectomized rats. The decrease of percentages of purified VN in partial hepatectomized rat was observed at 24 h. The changes of VN in both electrophoretic behavior and purified amount were so remarkable at 24 h after partial hepatectomy that VNs at 24 h after operation were further analyzed.

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, 1977Go). 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., 1995Go), DQESXK, where X is predicted to be cysteine according to the cDNA sequence.


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Table I. Amino acid composition of rat VNs
 
Carbohydrate composition and isoelectric focusing
As summarized in Table II, postoperative VNs were found to have markedly decreased carbohydrate sizes: 13 kDa, 6 kDa, and 4 kDa for VNs of nonoperated, sham-operated and partially hepatectomized rats, respectively. The decrease of total carbohydrates may account for the smaller molecular mass of the partially hepatectomized VN (Figure 1A, lane c). As estimated from the mannose concentrations (Table II), nonoperated rat VN is possibly glycosylated at all four potential N-glycosylation sites present in rat VN polypeptides as deduced from the cDNA sequence (Otter et al., 1995Go), while only one or two sites per molecule on average are N-glycosylated in VNs after partial hepatectomy and sham operation, respectively.


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Table II. Carbohydrate composition of rat VNs
 
After partial hepatectomy, the total carbohydrate concentration of VN was reduced compared with that after sham operation, with sialic acid, galactose, and mannose concentrations decreased to about half while GlcNAc concentration rather increased to twice that after sham operation. This may suggest an increased branching of N-linked oligosaccharides of the VN synthesized by partially hepatectomized rat liver, whereas the number of oligosaccharides transferred to the peptide decreased. The ratio of (Gal+GalNAc):NeuAc was about 1:1 for VN of nonoperated rats, while it was about 1:2 for VNs of sham-operated and partially hepatectomized rats, suggesting abnormal sialylation of postoperative rat VNs, i.e., that two sialic acids are linked to one (Gal or GalNAc) residue and/or sialic acids are linked to other residues such as branching GlcNAc.

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., 1993Go) 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., 1999Go). 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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Table III. Reactivity of rat VNs with HRP-lectins
 


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Fig. 2. Reactivities of HRP-lectins to VNs. VNs (0.1–0.2 µg/100 µl in TBS) were dot-blotted onto a PVDF membrane, blocked with 3% BSA and reacted with various HRP-lectins (10 µg/ml in TBS) at room temperature for 1 h. The bound HRP-lectins were developed with 4-chloro-1-naphthol/H2O2 as described in the text. The staining intensities were measured by a refractive densitometer at an absorbance of 370 nm for lectin staining or at 550 nm for CBB staining. (A) Reactivity with L-PHA, (B) reactivity with E-PHA, and (C) reactivity with PVL. Circle, VN from nonoperated rat plasma; square, VN from sham-operated rat plasma; and triangle, VN from partially hepatectomized rat plasma.

 
These VNs bind with SSA, an {alpha}2–6-linked sialic acid-specific lectin, but not MAM, an {alpha}2–3-linked sialic acid-specific lectin, indicating that there are sialic acids linked to galactose residue through {alpha}2–6 linkage but not {alpha}2–3 linkage, although further information about unusual sialyl substitutions could not be obtained from the lectin reactivities. The VNs reacted with PNA only after desialylation (Table III), indicating that these VNs commonly contain O-linked glycans with a sialylated Galß1–3GalNAc-Ser/Thr structure, which corresponds to the presence of GalNAc in these VNs (Table II), but the difference among O-linked glycans in these VNs could not be determined from the lectin reactivity. These results showed glycosylation of VN of partially hepatectomized rats had changed dramatically from those of nonoperated and sham-operated rats in both amount and structure of N-linked glycans.

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., 1996aGo,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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Fig. 3. Binding of purified VNs to type I collagen. Type I collagen (1 µg/100 ml) was coated onto wells of microtiter plates (Immulon 1), and then the wells were blocked with 5% BSA in TBS. Various concentrations of purified VNs (50 µl) were added to each well, followed by incubation for 1 h. After being washed with TBS three times, the amount of rat VN bound to immobilized collagen was measured with an HRP-conjugated rabbit anti-human VN IgG and an ELISA as described under Materials and methods. The absorbance of collagen-bound VN was corrected for the antibody reactivity of each VN. (A) The dose dependence of optical responses on the concentration of VN immobilized on the well. (B) Binding of the VNs to collagen. The absorbance of collagen-bound VN was corrected using the reactivity of each VN to rabbit anti-human VN IgG in Figure 3A. The symbol used in (A) and (B): circle, nonoperated rat VN; square, sham-operated rat VN; and triangle, partially hepatectomized rat VN. (C) The ratio of bound VNs of sham-operated rat (square) or partially hepatectomized rat (triangle) to bound VN of nonoperated rat.

 
Effect of glycosidase digestion on the binding activity to type I collagen
To determine the structural factor modulating the collagen binding, VN from nonoperated rat plasma was digested with neuraminidase or N-glycosidase F, and the effect of deglycosylation on collagen binding was assayed by ELISA. The raw absorbance data of bound VNs was corrected using the relative antibody reactivity of each VN by the same procedure as in Figure 3, and is presented in Figure 4A,B. As shown in Figure 4A, the reactivity of VN to collagen was considerably increased by desialylation and was calculated to be about 2.7–3.0 times that of control VN, while deN-glycosylation of rat VN by N-glycosidase F increased its collagen binding by only 1.0–1.3 times (Figure 4C). The effect of deN-glycosylation on collagen binding of VN was small compared to that of desialylation. One possible reason is that deN-glycosylation leaves an O-linked sugar chain, which is revealed to contain some sialic acids by the reactivity with PNA, on the peptide (Table III). Another is that the negative charges introduced into the peptide by N-glycosidase F treatment may interfere with the collagen binding, because the N-linked Asn residue is converted to an Asp residue after hydrolysis with N-glycosidase F. We tried to remove O-glycan with O-glycanase after sialidase treatment of VN, but O-glycan in rat VN was resistant to the O-glycanase digestion.



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Fig. 4. Binding of desialylated VN and deN-glycosylated VN to type I collagen immobilized on plastic plates. Nonoperated rat VN was enzymatically desialylated or de N-glycosylated and run for the ELISA. The absorbance of collagen bound VN was corrected for the antibody reactivity of each VN. (A) Binding of desialylated VN. (B) Binding of deN-glycosylated VN to type I collagen. The symbols used in (A): solid square, desialylated VN; open square, untreated VN; and in (B): solid triangle, deN-glycosylated VN; open triangle, untreated VN. (C) The ratio of bound glycosidase-treated VNs to that of untreated VN. Solid square, Desialylated VN; and solid triangle, deN-glycosylated VN.

 
These findings indicate that glycans covalently linked to VN, especially sialic acid residues located at the non-reducing end of the oligosaccharides, interfere with the collagen binding of VN. Therefore the increased binding of partially hepatectomized rat VN to collagen is, at least in part, related to changes of the glycosylations of the VN.


    Discussion
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Previously we showed that glycosylation of human and chicken VN contributes to its resistance to protease (Uchibori et al., 1992Go). The novel function of the glycosylation of VN in relation to the interaction between VN and collagen was elucidated in this study. The major findings are that the VN synthesized by rat livers at 24 h after partial hepatectomy exhibited markedly enhanced binding to type I collagen compared to nonoperated and sham-operated rat VN (Figure 3), and that the changes in glycosylation occurred concomitantly (Tables II and III, Figure 2). Enzymatic deglycosylation of VN showed that the glycosylation, especially sialylation of VN, modulates its collagen binding activity (Figure 4). Therefore, the changes of collagen binding of postoperative VNs are considered to be related to the alterations of glycosylation during liver regeneration.

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., 1985Go), which also causes the decrease of total glycoprotein synthesis in regenerating liver (Okamoto and Akamatsu, 1977Go). 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., 1995Go), and I (Okamoto and Akamatsu, 1978Go), galactosyltransferase (Bauer et al., 1976Go) and sialyltransferase (Ip, 1979Go; Serafini-Cessi, 1977Go). 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., 1996Go). In our previous study, the collagen binding domains of VN were identified as hemopexin II and I domains (Yoneda et al., 1998Go). 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, 1991Go), 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, 1991Go). 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., 1965Go; Bekesi and Winzler, 1967Go) and that partial hepatectomy causes a transient decrease in the total amount of plasma proteins (Bresnick, 1971Go). 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., 1997Go), 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., 1998Go).

Recent evidence indicates that VN may be involved in wound healing or tissue remodeling processes associated with injury or inflammation (Inuzuka et al., 1994Go; Seiffert, 1997Go; Preissner and Seiffert, 1998Go). 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., 1995Go, 1996; Seiffert, 1997Go). 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., 1987Go; Inuzuka et al., 1992Go; Kobayashi et al., 1994Go), while the collagen-binding VN in plasma increases in these diseases, compared with those of healthy controls (Inuzuka et al., 1992Go; Yamada et al., 1996Go), and the amount of collagen-binding VN was significantly correlated with certain fibrous markers (Yamada et al., 1996Go). The co-deposition of VN with collagen fibrils has been observed in hepatic fibrosis (Kobayashi et al., 1994Go) and cirrhosis (Inuzuka et al., 1992Go), 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., 1994Go). 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.


    Materials and methods
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 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Materials
Horseradish peroxidase (HRP)-labeled rabbit IgG against human-VN, unconjugated rabbit anti-human VN IgG and HRP-conjugated sheep anti-rabbit IgGs were purchased from The Binding Site Ltd. (Birmingham, England). Various HRP-lectins, Concanavalin A (ConA), Lens culinaris agglutinin (LCA), Phaseolus vulgaris erythroagglutinin (E-PHA), Ricinus communis agglutinin (RCA), peanut agglutinin (PNA), soybean agglutinin (SBA), Sambucus sieboldiana agglutinin (SSA), Maackia amurensis mitogen (MAM) and Ulex europeus agglutinin (UEA-1) were purchased from Seikagaku Kogyo (Tokyo, Japan), and biotin-labeled Psathyrella velutina lectin (PVL) was prepared in our laboratory (Ueda et al., 1999Go). Molecular weight standard and isoelectric focusing kits were purchased from Dai-ichi Kagaku Yakuhin (Tokyo, Japan). PVDF membrane (Immobilon P; pore size 0.45 µm) was purchased from Millipore Corp. (Bedford, MA). N-Glycosidase F (PNGase F, Flavobacterium meningosepticum) and neuraminidase (Vibrio cholerae) were purchased from Boehringer Mannheim (Mannheim, Germany). Type I collagen from calfskin and other reagents were special grade from Wako Pure Chemicals (Osaka, Japan).

Animals
Male Wistar rats aged 5–6 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 (6:00–18:00) and darkness. Two-thirds partial hepatectomy was performed under diethyl ether anesthesia as reported previously (Oda-Tamai et al., 1985Go). 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., 1988Go). 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., 1996aGo).

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, 1985Go). The amount of total protein in plasma was determined as described previously (Oda-Tamai et al., 1985Go).

SDS–PAGE and isoelectric focusing
SDS–PAGE was carried out using 9% polyacrylamide gel according to the method of Laemmli (Laemmli, 1970Go) under reducing conditions. Isoelectric focusing was performed according to the manufacturer’s 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 manufacturer’s instructions (Jusco Co., Tokyo, Japan). The index of composition divergence (D) was calculated according to Black and Harkins (Black and Harkins, 1977Go) using the equation, D = [{Sigma}(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., 1990Go). Sialic acid component analysis was performed according to the method of Hara et al. (1987)Go 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 0.1–0.2 µg/100 µl and reacted with HRP-lectins as previously described (Kitagaki-Ogawa et al., 1990Go). 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., 1986Go; Yoneda et al., 1998Go). 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 Tris–HCl 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 (0.0018–0.13 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 SDS–PAGE and the loss of reactivity with biotin-PVL or HRP-Con A, respectively, on Western blotting.


    Acknowledgments
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
This research was supported in part by Grants-in-Aid for Scientific Research on Priority Areas No. 11121213, and for Scientific Research (C) No. 09680585 from the ministry of Education, Science, Sports, and Culture, and the Naito Foundation for Scientific Research (HO). We thank Dr. Takemi Yatohgo of Itoham Foods Inc. for helpful suggestions, and Prof. Isamu Matsumoto and Prof. Masako Ohashi, Ochanomizu University for encouragement and support in densitometry.


    Abbreviations
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
VN, vitronectin; HRP, horseradish peroxidase; ConA, Concanavalin A; LCA, Lens culinaris agglutinin; E-PHA, Phaseolus vulgaris erythroagglutinin; RCA, Ricinus communis agglutinin; PNA, peanut agglutinin; SBA, soybean agglutinin; SSA, Sambucus sieboldiana agglutinin; MAM, Maackia amurensis mitogen; PVL, Psathyrella velutina lectin; UEA-I, Ulex europeus agglutinin I; DABS, 4-[4-(dimethylamino)phenylazo]benzenesulfonyl-chloride; TBS, 10 mM Tris–HCl buffer (pH 7.5) containing 140 mM NaCl.


    Footnotes
 
1 Present address: Biosignaling Department, National Institute of Bioscience and Human Technology, 1–1 Higashi, Tsukuba, Ibaraki, Japan Back

2 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 Abbreviations
 References
 
Bauer,C.H., Hassels,B.F. and Reutter,W.G. (1976) Galactose metabolism in regenerating rat liver. Biochem. J., 154, 141–147.[ISI][Medline]

Bekesi,J.G.and Winzler,R.J. (1967) The metabolism of plasma glycoproteins. Studies on the incorporation of L-fucose-1-14-C into tissue and serum in the normal rat. J. Biol. Chem., 242, 3873–3879.

Black,J.A.and Harkins,R.N. (1977) Amino acid compositions and evolutionary relationships with protein families. J. Theor. Biol., 66, 281–295.[ISI][Medline]

Blithe,D.L. (1993) Biological functions of oligosaccharides on glycoproteins. Trends Glycosci. Glycotechnol., 5, 81–98.

Bresnick,E. (1971) Regenerating liver: An experimental model for the study of growth. In Busch,H. (ed.), Methods in Cancer Research, chapter 8. Academic Press, New York, pp. 347–397.

Dahlback,B. and Podack,E.R. (1985) Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group. Biochemistry, 24, 2368–2374.[ISI][Medline]

Gebb,C., Hayman,E.G., Engvall,E. and Ruoslahti,E. (1986) Interaction of vitronectin with collagen. J. Biol. Chem., 261, 16698–16703.[Abstract/Free Full Text]

Hara,S., Takemori,Y., Yamaguchi,M., Nakamura,M. and Ohkura,Y. (1987) Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal. Biochem., 164, 138–145.[ISI][Medline]

Hughes,R.C. (1997) Adhesive glycoproteins and receptors. In Montreuil,J., Vliegenthart,J.F.G., and Schachter,H. (eds.), Secondary Adhesive Glycoproteins and Receptors. Elsevier Science B.V., New York, pp. 547–570.

Inuzuka,S., Ueno,T. and Tanikawa,K. (1994) Fibrogenesis in acute liver injuries. Path. Res. Pract., 190, 903–909.[ISI][Medline]

Inuzuka,S., Ueno,T., Torimura,T., Tamaki,S., Sakata,R., Sata,M., Toshida,H. and Tanikawa,K. (1992) VN in liver disorders: biochemical and immunohistochemical studies. Hepatology, 15, 629–636.[ISI][Medline]

Ip,C. (1979) Effect of partial hepatectomy and hydrocortisone administration on liver and serum sialyltransferase activities. Biochim. Biophys. Acta, 583, 14–19.[ISI][Medline]

Ishii,I., Takahashi,N., Kato,S., Akamatsu,N. and Kawazoe,Y. (1985) High-performance liquid chromatography analysis of changes of asparagine-linked oligosaccharides in regenerating rat liver. J. Chromatogr., 345, 134–139.[Medline]

Ishikawa-Sakurai,M. and Hayashi,M. (1993) Two collage N-binding domains of vitronectin. Cell. Struct. Funct., 18, 253–259.[ISI][Medline]

Izumi,M., Shimo-Oka,T., Morishita,N., Li,I. and Hayashi,M. (1988) Identification of the collagen-binding domain of vitronectin using monoclonal antibodies. Cell. Struct. Funct., 13, 217–225.[ISI][Medline]

Kato,S. and Akamatsu,N. (1984) Alterations in N-linked oligosaccharides of glycoproteins during rat liver regeneration. Biochim. Biophys. Acta, 798, 68–77.[ISI][Medline]

Kato,S. and Akamatsu,N. (1985) Alterations in fucosyl oligosaccharides of glycoproteins during rat liver regeneration. Biochem. J., 229, 521–528.[ISI][Medline]

Kato,S., Otsu,K., Ohtake,K., Kimura,Y., Yashiro,T., Suzuki,T. and Akamatsu,N. (1992) Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration. Exp. Cell Res., 198, 59–68.[ISI][Medline]

Kelm,R.J.J. and Mann,K.G. (1991) The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. J. Biol. Chem., 266, 9632–9639.[Abstract/Free Full Text]

Kemkes-Matthes,B., Preissner,K.T., Langenscheidt,F., Matthes,K.J. and Muller-Berghaus,G. (1987) S protein/vitronectin in chronic liver diseases: correlations with serum cholinesterase, coagulation factor X and complement component C3. Eur. J. Haematol., 39, 161–165.[ISI][Medline]

Kim,T.-H., Mars,W.M., Stolz,D.B., Petersen,B.E. and Michalopoulos,G.K. (1997) Extracellular matrix remodeling at the early stages of liver regeneration in the rat. Hepatology, 26, 896–904.[ISI][Medline]

Kitagaki-Ogawa,H., Yatohgo,T., Izumi,M., Hayashi,M., Kashiwagi,H., Matsumoto,I. and Seno,N. (1990) Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains. Biochim. Biophys. Acta, 1033, 49–56.[ISI][Medline]

Kobayashi,J., Yamada,S. and Kawasaki,H. (1994) Distribution of vitronectin in plasma and liver tissue: relationship to chronic liver disease. Hepatology, 20, 1412–1417.[ISI][Medline]

Laemmli,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.[ISI][Medline]

Macbeth,R.A.L., Bekesi,J.G., Sugden,E. and Bice,S. (1965) The metabolism of plasma glycoproteins. J. Biol. Chem., 240, 3707–3713.[Free Full Text]

McKeown-Longo,P.J. and Panetti,T.S. (1996) Structure and runction of vitronectin. Trends Glycosci. Glycotechnol., 8, 327–340.[ISI]

Miyoshi,E., Ihara,Y., Nishikawa,A., Sato,H., Uozumi,N., Hayashi,N., Fusamoto,H., Kamada,T. and Taniguchi,N. (1995) Gene expression of N-acetylglucosaminyltransferase III and V: a possible role for liver regeneration. Hepatology, 22, 1847–1855.[ISI][Medline]

Nakashima,N., Miyazaki,K., Ishikawa,M., Yothigo,T., Ogawa,H., Uchibori,H., Matsumoto,I., Seno,N. and Hayashi,M. (1992) Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide. Biochim. Biophys. Acta, 1120, 1–10.[ISI][Medline]

Oda-Tamai,S., Kato,S., Hara,S. and Akamatsu,N. (1985) Decreased transfer of oligosaccharide from oligosaccharide-lipid to protein acceptors in regenerating rat liver. J. Biol. Chem., 260, 57–63.[Abstract/Free Full Text]

Okamoto,Y. and Akamatsu,N. (1977) Synthesis in vitro of glycoprotein in regenerating rat liver. Biochim. Biophys. Acta, 498, 272–281.[ISI][Medline]

Okamoto,Y. and Akamatsu,N. (1978) UDP-N-acetylglucosamine-glycoprotein N-acetylglucosaminyltransferase in regenerating rat liver. Biochim. Biophys. Acta, 542, 21–27.[ISI][Medline]

Otter,M., Kiper,J., Rijken,D. and Zonneveld,A.J. (1995) hepatocellular localization of biosynthesis of vitronectin. Characterisation of the primary structure of rat vitronectin. Biochem. Mol. Biol. Int., 37, 563–572.[ISI][Medline]

Preissner,K.T. (1991) Structure and biological role of vitronectin. Annu. Rev. Cell. Biol., 7, 275–310.[ISI]

Preissner,K.T. and Seiffert,D. (1998) Role of vitronectin and its receptors in haemostasis and vascular remodeling. Thrombosis Res., 89, 1–21.[ISI][Medline]

Seiffert,D. (1997) Constitutive and regulated expression of vitronectin. Histol. Histopathol., 12, 787–797.[ISI][Medline]

Seiffert,D., Geisterfer,M., Gauldie,J., Young,E. and Podor,T.J. (1995) IL-6 stimulates vitronectin gene expression in vivo. J. Immunol., 155, 3180–3185.[Abstract]

Seiffert,D., Curriden,S.A., Jenne,D., Binder,B.R. and Loskutoff,D.J. (1998) Differential regulation of vitronectin in mice and humans in vitro. J. Biol. Chem., 271, 5474–5480.[Abstract/Free Full Text]

Serafini-Cessi,F. (1977) Sialyltransferase activity in regenerating rat liver. Biochem. J. 166, 381–386.[ISI][Medline]

Suzuki,J., Kondo,A., Kato,I., Hase,S. and Ikenaka,T. (1990) Improved method for fluorescence labeling of sugar chains with sialic acid residues. Agric. Biol. Chem., 55, 283–284.

Tomasini,B.R. and Mosher,D.F. (1990) Vitronectin. In Coller,B.S. (ed.), Progress in Hemostasis and Thrombosis. Saunders, Philadelphia/Sydney, pp. 269–305.

Uchibori,H., Ogawa,H., Matsumoto,I. and Seno,N. (1992) Contribution of the sugar chains to the stability for proteolysis of cell adhesive glycoprotein, vitronectin. Connective Tissue, 23, 117–124.

Ueda,H., Kojima,K., Saitoh,T. and Ogawa,H. (1999) Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid. FEBS Lett., 448, 75–80.[ISI][Medline]

Varki,A. (1993) Biological roles of oligosaccharides: all the theories are correct. Glycobiology, 3, 97–130.[Abstract]

Yamada,S., Kobayashi,J., Murawaki,Y., Suou,T. and Kawasaki,H. (1996) Collagen-binding activity of plasma vitronectin in chronic liver disease. Clin. Chim. Acta, 252, 95–103.[ISI][Medline]

Yatohgo,T., Izumi,M., Kashiwagi,H. and Hayashi,M. (1988) Purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct. Funct., 13, 281–292.[ISI][Medline]

Yoneda,A., Ogawa,H., Matsumoto,I., Ishizuka,I., Hase,S. and Seno,N. (1993) Structures of N-linked oligosaccharides on porcine plasma vitronectin. Eur. J. Biochem., 218, 797–806.[Abstract]

Yoneda,A., Kojima,K., Yamamoto,K., Matsumoto,I. and Ogawa,H. (1996) Porcine vitronectin, the most compact form of single-chain vitronectin. J. Biochem. (Tokyo), 120, 954–960.[Abstract]

Yoneda,A., Ogawa,H., Kojima,K. and Matsumoto,I. (1998) Characterization of the ligand binding activities of vitronectin. Specifications toward various lipids and identification of binding domains for lipids and other ligands using recombinant domains. Biochemistry, 37, 6351–6360.[ISI][Medline]

Zhuang,P., Blackburn,M.N. and C. Peterson. (1996a) Characterization of the denaturation and renaturation of human plasma vitronectin. I Biophysical characterization of protein unfolding and multimerization. J. Biol. Chem., 271, 14323–14332.[Abstract/Free Full Text]

Zhuang,P., Li,H., Williams,J.G., Wagner,N.V., Seiffert,D. and Peterson,C.B. (1996b) Characterization of the denaturationn and renaturation of human plasma vitronectin. II Investigation and the mechanism of formation of multimers. J. Biol. Chem., 271, 14333–14343.[Abstract/Free Full Text]