**2. Modulation of vitronectin activities by glycosylation**

De-*N*-glycosylation of plasma vitronectin by enzyme treatment significantly attenuated the cholesterol sulfate-binding activity while it increased the collagen-binding activity. De-*O*glycosylation or desialylation of vitronectin contributed to the stability for proteolysis (Uchibori, H., Ogawa, H., et al. 1992). These findings suggest that glycosylations modulate the ligand binding activities and the half-life of vitronectin *in vivo*. We prepared various recombinant domains of human vitronectin and mutants with certain domains deleted and expressed them separately in *E. coli* as fusion proteins. Using these recombinants, sulfatide-, phosphatidylserine-, cholesterol 3-sulfate-, type I collagen-, heparin-, and beta-endorphinbinding activities were found to be attributable to hemopexin domains 2 and 1. The possibility was suggested that the presence of a somatomedin B domain and/or connecting region flanking hemopexin domain 1 inactivated its heparin binding. Further, it was indicated that some of the ligand binding activities were modulated by glycosylation of plasma vitronectin, which enables modulation of its biological activities by a change in glycosylation accompanying the physiological or pathological state of the liver.

### **2.1 Changes in vitronectin during liver regeneration**

#### **2.1.1 Changes in collagen-binding activity of plasma vitronectin during early stage of liver regeneration**

In this study, we used the liver regeneration of rats induced by two-thirds partial hepatectomy as a model system to study whether and how vitronectin plays a role in tissue remodeling after hepatectomy and how glycosylations are involved in the physiological processes. Vitronectin was purified from plasma at different times during the liver regeneration process and analyzed by SDS-PAGE (Uchibori-Iwaki, H., et al. 2000). As shown in Fig. 2A, each vitronectin showed one band on SDS-PAGE, and 24 h after partial hepatectomy (PH-VN) plasma vitronectin had shifted to a low migration position compared to vitronectins purified from plasma of non-operated (NO-VN) or partially hepatectomized rats and sham-operated rats (SH-VN), suggesting that the molecular mass of vitronectin had shrunk to 65 kDa at 24 h after partial hepatectomy from the 68-69 kDa of other vitronectins. At 24 h after operation, the yield of PH-VN had decreased to 1/3 that of sham-operated rats, and it was restored by 240 h, when liver regeneration was completed. At this time point, the amino acid composition did not change significantly, and the composition divergence (Black, J.A. and Harkins, R.N. 1977) of PH-VN was 0.040 when taking that of SH-VN as 0.0. All three vitronectins had the same N-terminal sequence, indicating that the three vitronectins had high homology among the primary sequence (Uchibori-Iwaki, H., et al. 2000).

As shown in Fig. 2B, the purified vitronectins bound to type I collagen by ELISA in a concentration-dependent manner, and PH-VN was found to exhibit much greater binding to collagen, about 3 times higher than that of SH-VN and NO-VN (Uchibori-Iwaki, H., et al. 2000). The enhanced binding of PH-VN to immobilized collagen shown by ELISA was supported by surface plasmon resonance (SPR), as shown in Fig. 2C. The relative affinity per monomer of PH-VN is remarkably high compared with those of NO- and SH-VN, especially at the lower concentrations (Sano, K., et al. 2007).

De-*N*-glycosylation of plasma vitronectin by enzyme treatment significantly attenuated the cholesterol sulfate-binding activity while it increased the collagen-binding activity. De-*O*glycosylation or desialylation of vitronectin contributed to the stability for proteolysis (Uchibori, H., Ogawa, H., et al. 1992). These findings suggest that glycosylations modulate the ligand binding activities and the half-life of vitronectin *in vivo*. We prepared various recombinant domains of human vitronectin and mutants with certain domains deleted and expressed them separately in *E. coli* as fusion proteins. Using these recombinants, sulfatide-, phosphatidylserine-, cholesterol 3-sulfate-, type I collagen-, heparin-, and beta-endorphinbinding activities were found to be attributable to hemopexin domains 2 and 1. The possibility was suggested that the presence of a somatomedin B domain and/or connecting region flanking hemopexin domain 1 inactivated its heparin binding. Further, it was indicated that some of the ligand binding activities were modulated by glycosylation of plasma vitronectin, which enables modulation of its biological activities by a change in

glycosylation accompanying the physiological or pathological state of the liver.

**2.1.1 Changes in collagen-binding activity of plasma vitronectin during early stage of** 

In this study, we used the liver regeneration of rats induced by two-thirds partial hepatectomy as a model system to study whether and how vitronectin plays a role in tissue remodeling after hepatectomy and how glycosylations are involved in the physiological processes. Vitronectin was purified from plasma at different times during the liver regeneration process and analyzed by SDS-PAGE (Uchibori-Iwaki, H., et al. 2000). As shown in Fig. 2A, each vitronectin showed one band on SDS-PAGE, and 24 h after partial hepatectomy (PH-VN) plasma vitronectin had shifted to a low migration position compared to vitronectins purified from plasma of non-operated (NO-VN) or partially hepatectomized rats and sham-operated rats (SH-VN), suggesting that the molecular mass of vitronectin had shrunk to 65 kDa at 24 h after partial hepatectomy from the 68-69 kDa of other vitronectins. At 24 h after operation, the yield of PH-VN had decreased to 1/3 that of sham-operated rats, and it was restored by 240 h, when liver regeneration was completed. At this time point, the amino acid composition did not change significantly, and the composition divergence (Black, J.A. and Harkins, R.N. 1977) of PH-VN was 0.040 when taking that of SH-VN as 0.0. All three vitronectins had the same N-terminal sequence, indicating that the three vitronectins had high homology among the primary sequence (Uchibori-Iwaki, H., et al.

As shown in Fig. 2B, the purified vitronectins bound to type I collagen by ELISA in a concentration-dependent manner, and PH-VN was found to exhibit much greater binding to collagen, about 3 times higher than that of SH-VN and NO-VN (Uchibori-Iwaki, H., et al. 2000). The enhanced binding of PH-VN to immobilized collagen shown by ELISA was supported by surface plasmon resonance (SPR), as shown in Fig. 2C. The relative affinity per monomer of PH-VN is remarkably high compared with those of NO- and SH-VN, especially

**2.1 Changes in vitronectin during liver regeneration** 

at the lower concentrations (Sano, K., et al. 2007).

**liver regeneration** 

2000).

**2. Modulation of vitronectin activities by glycosylation** 

Fig. 2. Changes in electrophoretic mobility and collagen-binding of rat vitronectin at 24 h after partial hepatectomy. (A) SDS-PAGE of vitronectins from non-operated rats (N), partially hepatectomized (PH) rats at 6-240h after operation, and sham-operated (SH) rats at 24 h after operation. (B) Type I collagen (1 μg/100 μL) was coated onto wells of microtiter plates. After blocking with 5% BSA, various concentrations of purified vitronectins were added to each well. The bound vitronectin was measured using HRP-conjugated rabbit antihuman vitronectin IgGs and ELISA. The absorbance of collagen-bound vitronectin was corrected for the antibody reactivity of each vitronectin. (C) Collagen was immobilized on a CM5 sensor chip, and each vitronectin in PBS was injected onto the sensor chip at a flow rate of 20 μL/min at 20°C. The change of resonance units (RU) was corrected by subtracting the value on the BSA-immobilized reference cell.

#### **2.1.2 Changes in glycosylation and carbohydrate concentration of vitronectin during early stage of liver regeneration**

As shown in Fig. 3, the carbohydrate analyses of the three vitronectins indicated that total carbohydrate contents of PH-VN and SH-VN decreased to one-third and one-half of that of NO-VN, respectively, and that a remarkable decrease in sialic acids and amounts of glycans occurred due to partial hepatectomy. The lectin reactivity of the three vitronectins indicated that these vitronectins contain complex-type *N*-linked oligosaccharides. The reactivity toward *Phaseolus vulgaris* lectin L4 (L-PHA) varied remarkably among vitronectins, and PH-VN showed marked reactivity with L-PHA, but SH- and NO-VNs reacted only slightly, suggesting that tri- or tetraantennary lactosamine-branched structures multiplied dramatically after partial hepatectomy. The specificity of PVL toward clustered sialyl residues (Ueda, H., Kojima, K., et al. 1999) (Ueda, H., Matsumoto, H., et al. 2002), the

Matrix Restructuring During Liver Regeneration

PH-VNs.

enzyme.

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 85

which were cross-linked by disulfide-bonds, as measured by the intensity ratio of bands in SDS-PAGE before and after reduction, as shown in Fig. 4C. The enhanced collagen-binding activity of PH-VN was attributable to a multivalent effect that was due to the increase in the sizes and amounts of multimer vitronectins. The increase in multimer vitronectins in active form in various ligand-binding activities will accelerate the matrix incorporation of

Fig. 4. The collagen binding activities (A), molecular weight and multimerization (B), and relative amounts of multimer (white bar) to monomer (black bar) of glycan-trimmed human vitronectin. The typical complex-type glycan structures of mammalian vitronectin was sequentially trimmed by sialidase (S), β-galactosidase (G), β-hexosaminidase (H), and *N*glycosidase F (NG). U: untreated vitronectin; C: control vitronectin incubated without

**2.1.4 Effects of glycosylation of vitronectin on hepatic stellate cell spreading** 

Hepatic stellate cells are fibrotic cells that are induced during hepatic inflammation and are the major source of the newly synthesized ECM during hepatic fibrosis, whereas the survival or apoptosis of hepatic stellate cells is critical for the development or resolution, respectively, of liver fibrosis in chronic liver diseases (Benyon, R.C. and Arthur, M.J. 2001). In the normal liver, hepatic stellate cells have a low proliferation rate and produce trace amounts of ECM. As liver fibrosis progresses, hepatic stellate cells proliferate, but during

remarkably decreased reactivity of PH-VN with *Psathyrella velutina* lectin (PVL), together with the decrease in the reactivities with concanavalin A (Con A) and *Lens culinaris* lectin (LCA), indicate that the sialylated *N-*glycans markedly decreased after partial hepatectomy, which agrees well with the decreased amounts of carbohydrates including sialyl residues of the PH-VN. The changes in branching glycans would be attributable to the activity of several glycosyltransferases, which have been reported to increase (Miyoshi, E. , et al. 1995; Okamoto, Y., et al. 1978), while the decreased *N*-glycosylation of vitronectin at 24 h after partial hepatectomy could be attributed to the attenuation of the oligosaccharide transferase activity in microsomes (Oda-Tamai, S., et al. 1985).

Fig. 3. The carbohydrate concentration, composition, and reactivity with lectins of vitronectins (VN) from non-operated (NO), sham-operated (SH), and partially hepatectomized (PH) rats.

#### **2.1.3 Mechanism of enhanced collagen binding by change of vitronectin glycosylation**

To study the enhancement of the mechanism for collagen binding, NO-VN was deglycosylated by sequential exoglycosidase treatments and collagen binding activity was analyzed by ELISA. As shown in Fig. 4A, collagen-binding of vitronectin gradually increased with step-wise trimming of glycans. Deglycosylated vitronectin (NG) showed collagen-binding activity three times higher than that of control vitronectin, suggesting that the enhancement of collagen binding of PH-VN is due to the changes in glycosylation (Sano, K., et al. 2007).

The deglycosylated NO-VNs were analyzed for multimer formation by ultracentrifugation, and the multimer sizes were calculated from the weight average molecular weight of vitronectin (Fig. 4B). The multimer sizes were gradually increased by step-wise deglycosylation, accompanied with an increase of the amounts of multimer vitronectins,

remarkably decreased reactivity of PH-VN with *Psathyrella velutina* lectin (PVL), together with the decrease in the reactivities with concanavalin A (Con A) and *Lens culinaris* lectin (LCA), indicate that the sialylated *N-*glycans markedly decreased after partial hepatectomy, which agrees well with the decreased amounts of carbohydrates including sialyl residues of the PH-VN. The changes in branching glycans would be attributable to the activity of several glycosyltransferases, which have been reported to increase (Miyoshi, E. , et al. 1995; Okamoto, Y., et al. 1978), while the decreased *N*-glycosylation of vitronectin at 24 h after partial hepatectomy could be attributed to the attenuation of the oligosaccharide transferase

Fig. 3. The carbohydrate concentration, composition, and reactivity with lectins of vitronectins (VN) from non-operated (NO), sham-operated (SH), and partially

**2.1.3 Mechanism of enhanced collagen binding by change of vitronectin glycosylation**  To study the enhancement of the mechanism for collagen binding, NO-VN was deglycosylated by sequential exoglycosidase treatments and collagen binding activity was analyzed by ELISA. As shown in Fig. 4A, collagen-binding of vitronectin gradually increased with step-wise trimming of glycans. Deglycosylated vitronectin (NG) showed collagen-binding activity three times higher than that of control vitronectin, suggesting that the enhancement of collagen binding of PH-VN is due to the changes in glycosylation (Sano,

The deglycosylated NO-VNs were analyzed for multimer formation by ultracentrifugation, and the multimer sizes were calculated from the weight average molecular weight of vitronectin (Fig. 4B). The multimer sizes were gradually increased by step-wise deglycosylation, accompanied with an increase of the amounts of multimer vitronectins,

activity in microsomes (Oda-Tamai, S., et al. 1985).

hepatectomized (PH) rats.

K., et al. 2007).

which were cross-linked by disulfide-bonds, as measured by the intensity ratio of bands in SDS-PAGE before and after reduction, as shown in Fig. 4C. The enhanced collagen-binding activity of PH-VN was attributable to a multivalent effect that was due to the increase in the sizes and amounts of multimer vitronectins. The increase in multimer vitronectins in active form in various ligand-binding activities will accelerate the matrix incorporation of PH-VNs.

Fig. 4. The collagen binding activities (A), molecular weight and multimerization (B), and relative amounts of multimer (white bar) to monomer (black bar) of glycan-trimmed human vitronectin. The typical complex-type glycan structures of mammalian vitronectin was sequentially trimmed by sialidase (S), β-galactosidase (G), β-hexosaminidase (H), and *N*glycosidase F (NG). U: untreated vitronectin; C: control vitronectin incubated without enzyme.

#### **2.1.4 Effects of glycosylation of vitronectin on hepatic stellate cell spreading**

Hepatic stellate cells are fibrotic cells that are induced during hepatic inflammation and are the major source of the newly synthesized ECM during hepatic fibrosis, whereas the survival or apoptosis of hepatic stellate cells is critical for the development or resolution, respectively, of liver fibrosis in chronic liver diseases (Benyon, R.C. and Arthur, M.J. 2001). In the normal liver, hepatic stellate cells have a low proliferation rate and produce trace amounts of ECM. As liver fibrosis progresses, hepatic stellate cells proliferate, but during

Matrix Restructuring During Liver Regeneration

decreased HSC spreading activity of PH-VN than *N*-glycans do.

**2.1.5 Site-specific glycosylations of rat vitronectin** 

type molecules were attached.

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 87

*N*-glycosidase F (PNGase F) treatment. In addition, PNGase F converts asparagine to aspartate, which may reduce the effect of the decrease of the negative charge of sialic acids. Because a clear difference between de-*N*-glycosylated and non-treated samples in cell spreading was still observed, suggesting the contribution of fibronectin-glycans to some extent, it cannot be concluded from this result that *O*-glycans contribute more to the

To address the effects of glycosylation of vitronectin on integrin-mediated signaling, the focal adhesion kinase (FAK) of HSCs was compared among NO-, SH-, and PH-VN. As shown in Fig. 5B, the amount of phosphorylated FAK on PH-VN was decreased in proportion to cell spreading. These results suggest that the change in vitronectin glycosylation due to partial hepatectomy is able to regulate activation of the integrinmediated signaling pathway. In addition, the effect of phosphorylated FAK on

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, Y. and Akamatsu, N. 1977). The contradictory decrease of total glycoprotein synthesis in regenerating rat liver is due to the attenuation of oligosaccharide transferase activity in microsomes (Oda-Tamai, S., et al. 1985). Alterations in the glycan structure of total hepatic glycoproteins have been also suggested during liver regeneration (Kato, S. and Akamatsu, N. 1985; Ishii, I., et al. 1985). However, the changes in the glycans of a particular glycoprotein have not been well characterized. For these reasons, we investigated the sitespecific glycosylations and changes after partial hepatectomy in rat plasma vitronectin.

Liquid chromatography/mass spectrometry analysis (LC/MSn) of Glu-C glycopeptides determined the site-specific glycosylation of each vitronectin. Four potential sites, Asn86, Asn96, Asn167, and Asn240 were revealed to be *N*-glycosylated, while the peptides of residues Thr110-Thr124 were *O*-glycosylated. The most frequent *N*-glycan structures site-specifically found for each site are shown in Fig. 6. At Asn86, Asn96, and Asn240, biantennary complextype trisialoglycans with or without core fucosylation and with different amounts of *O*acetylated NeuNAc were deduced (Fig. 6), whereas biantennary hybrid-type *N*-glycans were found to be the major structures at Asn167. In the Thr110–Thr124 region, the highly sialylated glycans were detected in the negative ion mode spectrum, and analysis in positive ion mode revealed that a Hex-HexNAc unit was located in the inner region of the glycans. From the results of lectin reactivity (*18*), it was inferred that one to three sialylated core-1

These *O*-glycans contained disialic acid, which was chemically confirmed by fluorometric C7/C9 analyses and mild acid hydrolysate-fluorometric anion-exchange chromatography (Yasukawa, Z., Sato, C., et al. 2005). PH-VN had less disialyl *O*-glycans and complex-type N-

At the same time, alterations in the glycosylation of fibronectin (FN) after PH were different from those of vitronectin. The carbohydrate concentration of PH-FN decreased to 66% of that of NO- and SH-FNs. LC/MSn revealed that eight kinds of complex-type *N*-glycan structures were present in NO-, SH-, and PH-FNs, and that bi- and trisialobiantennary

glycans, but more core-fucosylated *N*-glycans than NO-VN (Sano, K., et al. 2010).

neuraminidase-treated NO-VN was decreased to an extent similar to that on PH-VN.

the resolution of fibrosis there is extensive apoptosis that coincides with degradation of the liver scar (Benyon, R.C. and Arthur, M.J. 2001). It was reported that the activation of hepatic stellate cells increased the expression of integrin αvβ3, which is the major receptor of vitronectin on the cell surface, and promotes their proliferation and survival (Kato, S. and Akamatsu, N. 1985). We determined the structure and changes of rat vitronectin glycans during liver regeneration, and observed the relationship between the survival signaling of hepatic stellate cells and glycosylation of vitronectin.

Fig. 5. Spreading and FAK-phosphorylation of HSCs on vitronectins. HSCs were plated on substrates coated with 10 μg/mL of vitronectins purified from NO, SH, or PH rats or desialylated (S) or de-*N*-glycosylated (NG) vitronectin. After 90-min incubation at 37°C in 5% CO2, the % of the cells spread and FAK-phosphorylated were assessed by taking those on NO-VN as 1. Photomicrographs at ×40 magnification are shown on the upper panel. The data were analyzed by Student's *t-*test. The data represent the means S.D. (n=6); \*\*\*, p<0.001; \*, p<0.05 compared with that on NO-VN.

In this study, vitronectins were purified from rat plasma at 24 hours after partial hepatectomy, sham-operation, or non-operated and designated as PH-, SH-, and NO-VN, respectively. The effect of PH-VN on HSC spreading was decreased to 1/2 of that of NO- or SH-VN (Fig. 5A). HSC spreading was also decreased on neuraminidase-treated vitronectin compared with untreated vitronectin, whereas it was decreased less on de-*N-*glycosylated NO-VN. These results indicate the importance of glycosylation, particularly sialylation, of vitronectin in HSC spreading. The effect of de-*N*-glycosylation was small compared with that of desialylation, because many sialic acid residues still remained on the *O*-glycans after

the resolution of fibrosis there is extensive apoptosis that coincides with degradation of the liver scar (Benyon, R.C. and Arthur, M.J. 2001). It was reported that the activation of hepatic stellate cells increased the expression of integrin αvβ3, which is the major receptor of vitronectin on the cell surface, and promotes their proliferation and survival (Kato, S. and Akamatsu, N. 1985). We determined the structure and changes of rat vitronectin glycans during liver regeneration, and observed the relationship between the survival signaling of

Fig. 5. Spreading and FAK-phosphorylation of HSCs on vitronectins. HSCs were plated on substrates coated with 10 μg/mL of vitronectins purified from NO, SH, or PH rats or desialylated (S) or de-*N*-glycosylated (NG) vitronectin. After 90-min incubation at 37°C in 5% CO2, the % of the cells spread and FAK-phosphorylated were assessed by taking those on NO-VN as 1. Photomicrographs at ×40 magnification are shown on the upper panel. The data were analyzed by Student's *t-*test. The data represent the means S.D. (n=6); \*\*\*, p<0.001;

In this study, vitronectins were purified from rat plasma at 24 hours after partial hepatectomy, sham-operation, or non-operated and designated as PH-, SH-, and NO-VN, respectively. The effect of PH-VN on HSC spreading was decreased to 1/2 of that of NO- or SH-VN (Fig. 5A). HSC spreading was also decreased on neuraminidase-treated vitronectin compared with untreated vitronectin, whereas it was decreased less on de-*N-*glycosylated NO-VN. These results indicate the importance of glycosylation, particularly sialylation, of vitronectin in HSC spreading. The effect of de-*N*-glycosylation was small compared with that of desialylation, because many sialic acid residues still remained on the *O*-glycans after

hepatic stellate cells and glycosylation of vitronectin.

\*, p<0.05 compared with that on NO-VN.

*N*-glycosidase F (PNGase F) treatment. In addition, PNGase F converts asparagine to aspartate, which may reduce the effect of the decrease of the negative charge of sialic acids. Because a clear difference between de-*N*-glycosylated and non-treated samples in cell spreading was still observed, suggesting the contribution of fibronectin-glycans to some extent, it cannot be concluded from this result that *O*-glycans contribute more to the decreased HSC spreading activity of PH-VN than *N*-glycans do.

To address the effects of glycosylation of vitronectin on integrin-mediated signaling, the focal adhesion kinase (FAK) of HSCs was compared among NO-, SH-, and PH-VN. As shown in Fig. 5B, the amount of phosphorylated FAK on PH-VN was decreased in proportion to cell spreading. These results suggest that the change in vitronectin glycosylation due to partial hepatectomy is able to regulate activation of the integrinmediated signaling pathway. In addition, the effect of phosphorylated FAK on neuraminidase-treated NO-VN was decreased to an extent similar to that on PH-VN.
