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

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 type molecules were attached.

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

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

Matrix Restructuring During Liver Regeneration

Z., et al. 2006).

regeneration.

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 89

neuraminidase treatment, and only the more acidic component of pI 4.1 reacted with mAb S2–566, which specifically recognizes the Neu5Acα2,8Neu5Acα2,3Gal structure (Yasukawa,

Fig. 7. Two-dimensional PAGE and western blotting of vitronectins. The first electrophoresis

The results were also supported by the immunodetection of each vitronectin after 2D-PAGE where both hyper- and hyposilalylated molecules were present and the hypersialylation in PH-VN was markedly attenuated (Fig. 3). These analyses showed that the oligosialic acid on the *O*-glycan significantly affected the pI of the acidic partially hepatectomized vitronectin fraction. In addition to the decrease of oligosialyl epitopes in PH-VN, undersialylation of

The presence of disialic acid structures in some glycoproteins was previously described (Finne, J., et al. 1977), and neural cell adhesion molecules have been very thoroughly studied (Finne, J., Krusius, T., et al. 1977). Changes in expression of the oligosialic epitope on serum glycoproteins under inflammatory conditions were also reported (Yasukawa, Z., et al. 2005) (Yasukawa, Z., et al. 2007). The fact that the amounts of disialic acid structures in PH-VN were decreased compared with those in NO-VN indicates that the inflammation caused by partial hepatectomy reduces disialylation on vitronectin. The results indicate that the decreased sialylation plays a key role in regulating the function of vitronectin in liver

was isoelectric focusing. The second electrophoresis was SDS-PAGE under reducing conditions on 7.5% polyacrylamide gel, followed by blotting onto a polyvinylidene difluoride membrane and immunostaining using sheep anti-vitronectin IgGs (A–D) or the anti-oligosialic acid monoclonal antibody S2–566 (E), and HRP-conjugated secondary

antibodies. Membranes were developed with ECL-Plus.

both *N*- and *O*-glycans was found in the basic PH-VN fraction.

glycans were the major structures (Sano, K., et al. 2008). Hybrid-type *N*-glycans and disialyl *O*-glycans were not detected. These results indicate that the alterations in the glycosylations of fibronectin and vitronectin were significantly different in the early stage of liver regeneration and demonstrate that these glycoproteins play different biological roles in the promotion of tissue remodeling processes.

Fig. 6. Site-specific glycosylation of rat plasma vitronectin. The four glycosylation sites on rat plasma vitronectin and the glycan structures at each glycosylation site were determined by glycopeptide analyses using LC/MSn. The most frequent glycan structures that were present at each site are presented.

#### **2.1.6 Change in isoelectric points and oligosialylation of vitronectin**

Highly sialylated *O*-glycans, which have a diNeuAc structure and were markedly decreased in PH-VN, affect the isoelectric points of vitronectins. Immunostaining of vitronectins after two-dimensional PAGE showed that each vitronectin has two components, pI 4.0 and 5.7 in NO-VN, that both shifted to higher pI, pI 4.3 and 6.0 in SH-VN, and further to pI 4.6 and 6.0 in PH-VN (Fig. 7). The pI of NO-VN was converted to one basic point, pI 6, after

glycans were the major structures (Sano, K., et al. 2008). Hybrid-type *N*-glycans and disialyl *O*-glycans were not detected. These results indicate that the alterations in the glycosylations of fibronectin and vitronectin were significantly different in the early stage of liver regeneration and demonstrate that these glycoproteins play different biological roles in the

Fig. 6. Site-specific glycosylation of rat plasma vitronectin. The four glycosylation sites on rat plasma vitronectin and the glycan structures at each glycosylation site were determined by glycopeptide analyses using LC/MSn. The most frequent glycan structures that were

Highly sialylated *O*-glycans, which have a diNeuAc structure and were markedly decreased in PH-VN, affect the isoelectric points of vitronectins. Immunostaining of vitronectins after two-dimensional PAGE showed that each vitronectin has two components, pI 4.0 and 5.7 in NO-VN, that both shifted to higher pI, pI 4.3 and 6.0 in SH-VN, and further to pI 4.6 and 6.0 in PH-VN (Fig. 7). The pI of NO-VN was converted to one basic point, pI 6, after

**2.1.6 Change in isoelectric points and oligosialylation of vitronectin** 

promotion of tissue remodeling processes.

present at each site are presented.

neuraminidase treatment, and only the more acidic component of pI 4.1 reacted with mAb S2–566, which specifically recognizes the Neu5Acα2,8Neu5Acα2,3Gal structure (Yasukawa, Z., et al. 2006).

Fig. 7. Two-dimensional PAGE and western blotting of vitronectins. The first electrophoresis was isoelectric focusing. The second electrophoresis was SDS-PAGE under reducing conditions on 7.5% polyacrylamide gel, followed by blotting onto a polyvinylidene difluoride membrane and immunostaining using sheep anti-vitronectin IgGs (A–D) or the anti-oligosialic acid monoclonal antibody S2–566 (E), and HRP-conjugated secondary antibodies. Membranes were developed with ECL-Plus.

The results were also supported by the immunodetection of each vitronectin after 2D-PAGE where both hyper- and hyposilalylated molecules were present and the hypersialylation in PH-VN was markedly attenuated (Fig. 3). These analyses showed that the oligosialic acid on the *O*-glycan significantly affected the pI of the acidic partially hepatectomized vitronectin fraction. In addition to the decrease of oligosialyl epitopes in PH-VN, undersialylation of both *N*- and *O*-glycans was found in the basic PH-VN fraction.

The presence of disialic acid structures in some glycoproteins was previously described (Finne, J., et al. 1977), and neural cell adhesion molecules have been very thoroughly studied (Finne, J., Krusius, T., et al. 1977). Changes in expression of the oligosialic epitope on serum glycoproteins under inflammatory conditions were also reported (Yasukawa, Z., et al. 2005) (Yasukawa, Z., et al. 2007). The fact that the amounts of disialic acid structures in PH-VN were decreased compared with those in NO-VN indicates that the inflammation caused by partial hepatectomy reduces disialylation on vitronectin. The results indicate that the decreased sialylation plays a key role in regulating the function of vitronectin in liver regeneration.

Matrix Restructuring During Liver Regeneration

for a number of biological systems.

**3.1.1 Principle of chromatofocusing** 

glycoproteins during liver degeneration.

**3.1.2 Method** 

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 91

changes in sialylation and separation of the matrix glycoproteins according to their pI in small amounts. The strategy is technically feasible and is applicable to various glycoproteins

Chromatofocusing was originally a method of purification by separating proteins according to their pI. The pI of a protein is the pH at which the protein has zero surface charge. If a buffer, initially adjusted to the first pH, is run through an ion exchange column (see Fig. 8A) and followed by another buffer of a second pH, a pH gradient is formed in the column (Fig. 8B) (Sluyterman, L.A.A. E., O 1978; Sluyterman, L.A. and Wijdenes, J. 1978). If this pH gradient is used to elute proteins bound to the ion exchanger, the proteins in the column are eluted in the order of their pI. If the pH of the mobile phase around a protein is higher than the protein's pI, the protein has a positive charge. Therefore, it is dissociated from the anion exchange column and eluted from the column (Fig. 8A-C). If the pH of the mobile phase is lower, the protein has a negative charge and remains in the column (Shan, L. and Anderson, D.J. 2001). In this study, we applied chromatofocusing to detect the changes in sialylation of

Fig. 8. Proteins having different pI are separated from each other as they pass through the anion exchanger column. A sample containing proteins that are (pI = 7.0), (pI = 5.5), and (pI = 4.0) was applied to the column. (A) When the first buffer (pH 7.0) was poured into the column, the pH in the column is 7.0; therefore, rectangle proteins (pI >7.0) are eluted. Triangle and hexagon proteins (pI<7.0) are still bound to the column because they are negatively charged at pH 7.0. (B), When the second buffer (pH 4.0) was added, the pH in the column becomes lower, and this is halfway through the elution. (C), When the second buffer (pH 4.0) was continuously loaded, the pH in the column became pH 4.0 throughout the column, and then the hexagon proteins (pI=4.0) were eluted at the end of the elution.

Rat plasma samples were collected at 24 h, 48 h, 72 h, 5 d, or 7 d after two-thirds hepatectomy or sham operation and stored at -80°C until use. Each plasma sample (200 μg/100 μl) was applied to chromatofocusing using a Mono P5/50 GL column (GE

In contrast to PH-VN, the glycosylation and biological activities of vitronectin in cirrhotic plasma were differentially changed. Yamada's group reported that vitronectin, which is active in collagen-binding in plasma, increased and correlated with certain fibrous markers (Yamada, S., et al. 1997, Yamada, S., et al. 1996), that the concentration of vitronectin in liver tissue was significantly increased in chronic liver disease compared with that in normal controls, and that vitronectin was colocalized at fibrous sites (Kobayashi, J., et al. 1994). Several reports supported the observation; therefore, the immunoreactivity of vitronectin in liver can be considered a marker of chronic/mature fibrosis. The vitronectins in untreated plasma exist mainly in native inactive form and exhibit low collagen binding. Urea treatment of cirrhotic and normal plasma revealed that the ratio of active to inactive vitronectin in cirrhotic plasma increased to more than twice that in normal plasma, promoting the incorporation of vitronectin from plasma into the matrix proceeds in spite of the fact that the vitronectin concentration in cirrhotic plasma was only 70% of that in normal plasma (Suzuki, R., et al. 2001). It is important to elucidate the changes in the glycosylation and biological activities of vitronectin in cirrhotic plasma and compare them with those of liver regeneration.

#### **2.2 Summary**

The present study attempted to determine how alterations of glycans modulate the biological activities of vitronectin during the initial stage of liver regeneration. Plasma vitronectin was purified from partially hepatectomized and sham-operated rats at 24 hours after operation and from non-operated rats. We found that the glycosylation of vitronectin changed and decreased markedly after surgery. The multimer sizes of PH- and SH-VNs significantly increased compared with NO-VN, and the change was accompanied by an increase in collagen binding. It was indicated that these changes were due to the changes in glycosylation of vitronectin, especially decreased sialylation, which increased the size and amount of the multimers to enhance the collagen-binding activity by a multivalent effect. Adhesion and spreading of rat hepatic stellate cells on PH-VN was decreased to 1/2 of that on NO- or SH-VN. Similarly, desialylated NO-VN decreased the spreading of rat hepatic stellate cells to 1/2 of that of control vitronectin, indicating the importance of sialylation of vitronectin for activation of rat hepatic stellate cells. LC/MSn of vitronectin glycopeptides determined the site-specific glycosylation and the presence of highly sialylated *O*-glycans, which dramatically decreased after partial hepatectomy. Understanding the functional modulation of glycans on vitronectin may contribute to development of a strategy to regulate liver regeneration and matrix fibrosis in liver cirrhosis.

#### **3. New method to detect changes in sialylation**

As described in the previous sections, in the initial stage of the liver regeneration, alterations in sialylation of vitronectin modulate the important biological activities of vitronectin during tissue-remodeling processes by multiple steps. Like vitronectin, the sialylation of other glycoproteins changes under pathological conditions as well as during developmental stages, and altered sialylation often has significant implications in the physiological role of glycoproteins (Varki, A., Commings, R.D., Esko, J. D., Freeze, H. H.,Stanley, P., H., Bertozzi, C.R., Hart, G.W., Etzler, M.E. 2009)(Rutishauser, U. 1998). The aim of this section is to describe a fundamental method using chromatofocusing that enables detection of the changes in sialylation and separation of the matrix glycoproteins according to their pI in small amounts. The strategy is technically feasible and is applicable to various glycoproteins for a number of biological systems.

#### **3.1.1 Principle of chromatofocusing**

90 Liver Regeneration

In contrast to PH-VN, the glycosylation and biological activities of vitronectin in cirrhotic plasma were differentially changed. Yamada's group reported that vitronectin, which is active in collagen-binding in plasma, increased and correlated with certain fibrous markers (Yamada, S., et al. 1997, Yamada, S., et al. 1996), that the concentration of vitronectin in liver tissue was significantly increased in chronic liver disease compared with that in normal controls, and that vitronectin was colocalized at fibrous sites (Kobayashi, J., et al. 1994). Several reports supported the observation; therefore, the immunoreactivity of vitronectin in liver can be considered a marker of chronic/mature fibrosis. The vitronectins in untreated plasma exist mainly in native inactive form and exhibit low collagen binding. Urea treatment of cirrhotic and normal plasma revealed that the ratio of active to inactive vitronectin in cirrhotic plasma increased to more than twice that in normal plasma, promoting the incorporation of vitronectin from plasma into the matrix proceeds in spite of the fact that the vitronectin concentration in cirrhotic plasma was only 70% of that in normal plasma (Suzuki, R., et al. 2001). It is important to elucidate the changes in the glycosylation and biological activities of vitronectin in cirrhotic plasma and compare them with those of

The present study attempted to determine how alterations of glycans modulate the biological activities of vitronectin during the initial stage of liver regeneration. Plasma vitronectin was purified from partially hepatectomized and sham-operated rats at 24 hours after operation and from non-operated rats. We found that the glycosylation of vitronectin changed and decreased markedly after surgery. The multimer sizes of PH- and SH-VNs significantly increased compared with NO-VN, and the change was accompanied by an increase in collagen binding. It was indicated that these changes were due to the changes in glycosylation of vitronectin, especially decreased sialylation, which increased the size and amount of the multimers to enhance the collagen-binding activity by a multivalent effect. Adhesion and spreading of rat hepatic stellate cells on PH-VN was decreased to 1/2 of that on NO- or SH-VN. Similarly, desialylated NO-VN decreased the spreading of rat hepatic stellate cells to 1/2 of that of control vitronectin, indicating the importance of sialylation of vitronectin for activation of rat hepatic stellate cells. LC/MSn of vitronectin glycopeptides determined the site-specific glycosylation and the presence of highly sialylated *O*-glycans, which dramatically decreased after partial hepatectomy. Understanding the functional modulation of glycans on vitronectin may contribute to development of a strategy to

As described in the previous sections, in the initial stage of the liver regeneration, alterations in sialylation of vitronectin modulate the important biological activities of vitronectin during tissue-remodeling processes by multiple steps. Like vitronectin, the sialylation of other glycoproteins changes under pathological conditions as well as during developmental stages, and altered sialylation often has significant implications in the physiological role of glycoproteins (Varki, A., Commings, R.D., Esko, J. D., Freeze, H. H.,Stanley, P., H., Bertozzi, C.R., Hart, G.W., Etzler, M.E. 2009)(Rutishauser, U. 1998). The aim of this section is to describe a fundamental method using chromatofocusing that enables detection of the

regulate liver regeneration and matrix fibrosis in liver cirrhosis.

**3. New method to detect changes in sialylation** 

liver regeneration.

**2.2 Summary** 

Chromatofocusing was originally a method of purification by separating proteins according to their pI. The pI of a protein is the pH at which the protein has zero surface charge. If a buffer, initially adjusted to the first pH, is run through an ion exchange column (see Fig. 8A) and followed by another buffer of a second pH, a pH gradient is formed in the column (Fig. 8B) (Sluyterman, L.A.A. E., O 1978; Sluyterman, L.A. and Wijdenes, J. 1978). If this pH gradient is used to elute proteins bound to the ion exchanger, the proteins in the column are eluted in the order of their pI. If the pH of the mobile phase around a protein is higher than the protein's pI, the protein has a positive charge. Therefore, it is dissociated from the anion exchange column and eluted from the column (Fig. 8A-C). If the pH of the mobile phase is lower, the protein has a negative charge and remains in the column (Shan, L. and Anderson, D.J. 2001). In this study, we applied chromatofocusing to detect the changes in sialylation of glycoproteins during liver degeneration.

Fig. 8. Proteins having different pI are separated from each other as they pass through the anion exchanger column. A sample containing proteins that are (pI = 7.0), (pI = 5.5), and (pI = 4.0) was applied to the column. (A) When the first buffer (pH 7.0) was poured into the column, the pH in the column is 7.0; therefore, rectangle proteins (pI >7.0) are eluted. Triangle and hexagon proteins (pI<7.0) are still bound to the column because they are negatively charged at pH 7.0. (B), When the second buffer (pH 4.0) was added, the pH in the column becomes lower, and this is halfway through the elution. (C), When the second buffer (pH 4.0) was continuously loaded, the pH in the column became pH 4.0 throughout the column, and then the hexagon proteins (pI=4.0) were eluted at the end of the elution.

#### **3.1.2 Method**

Rat plasma samples were collected at 24 h, 48 h, 72 h, 5 d, or 7 d after two-thirds hepatectomy or sham operation and stored at -80°C until use. Each plasma sample (200 μg/100 μl) was applied to chromatofocusing using a Mono P5/50 GL column (GE

Matrix Restructuring During Liver Regeneration

7d had recovered to pH 4.7–4.5 (data not shown).

is Regulated by Glycosylation of the Matrix Glycoprotein Vitronectin 93

Rat plasma vitronectin at 24 h after partial hepatectomy was shifted to a higher pI than that of NO-VN on two-dimensional PAGE, as shown in Fig. 7. The result of chromatofocusing combined with immunodetection suggests that sialylation of vitronectin remained low with pI over pH 5 for the period from 24 to 72h after partial hepatectomy. The pI of PH-VN after

Fig. 10. The elution pattern of vitronectin with descending elution pH. The amount of vitronectin was measured by the band intensity of an immunoblot of the eluted fraction and is expressed as %, when taking that of 1 μg of purified NO-VN as 100%. Solid line: PH-VN

The elution pattern on chromatofocusing was very reproducible, and the pIs of NO-VN and PH-VN were found to be 4.7–4.5 and 5.2–4.8, respectively (Fig. 10). The immunostaining of PH-VN after two-dimensional PAGE (Fig. 7) indicated the presence of two components (pI 4.6 and 6.0), whereas PH-VN was eluted by chromatofocusing at intermediate pH, although the two methods agreed on the tendency for the pI of PH-VN to be considerably shifted toward alkalinity compared to that of NO-VN. This discrepancy may be due not only to the difference in the plasma sample lots but also because parts of the vitronectin eluted by chromatofocusing are multimerized in the buffer like the vitronectin in physiological plasma, which shows a broad pH range of elution peaks indicating variation in sialylation of the vitronectin molecules contained. Chromatofocusing has the advantage of analyzing the isoelectric property under physiological conditions, especially in detection of changes in sialylation of glycoproteins, and

the eluted fractions are utilizable for activity measurements (Kneba, M., et al. 1983).

Each plasma sample was subjected to chromatofocusing using FPLC, which can separate molecules by their isoelectric point (pI). Fractions eluted from the column were subjected to

(24 h), chain line: NO-VN, dotted line: pH of the eluted fraction.

**3.1.4 Discussion** 

**3.1.5 Summary** 

Healthcare Inc.) and a fast protein liquid chromatograph (FPLC; AKTA Purifier, GE Healthcare, Inc). The starting buffer was 0.025 M bis-Tris, pH 7.1, and the elution buffer was 10% Polybuffer74, pH 4.0. Buffers were filtered through 0.22 μm filters under vacuum and degassed. Samples were adjusted to the pH of the starting buffer, or exchanged by dialysis into the starting buffer. The column was pre-equilibrated with the starting buffer until the eluent was applied to the column, which was the same pH as that of starting buffer. After samples were applied to FPLC, the elution buffer was added. Samples were separated by the pI of each component protein in the column, and sequentially eluted from the column. The effluent was continuously monitored by absorbance at 280 nm and divided by 1 mL/tube. After the pH gradient ended, the column was washed with two column volumes of 2M NaCl to elute the molecules still bound to the column. Finally, the column was reequilibrated with five column volumes of starting buffer until UV absorbance and pH values reached a plateau. An aliquot (10 μL) of each fraction eluted from the column was subjected to SDS-PAGE on a 7% polyacrylamide gel, and the separated protein bands were electro transferred to a polyvinylidene difluoride membrane and detected with specific antibodies to vitronectin. Membranes were developed with ECL (GE Healthcare Inc.).

#### **3.1.3 Results**

An example of the elution pattern of rat plasma is shown in Fig. 9. As the buffer flowed through the chromatofocusing column, the pH decreased and a descending pH gradient was generated. As shown in Fig. 10, when vitronectin in rat plasma was separated by chromatofocusing, NO-VN was eluted at pH 4.7–4.5, while PH–VN (24 h) was eluted at pH 5.2-4.8.

Fig. 9. Chromatogram of non-operated rat plasma. Dotted line, pH; solid line, absorbance at 280 nm. Flow rate: 0.5 mL/min, temperature: 4°C. Other conditions are described in the text.

Rat plasma vitronectin at 24 h after partial hepatectomy was shifted to a higher pI than that of NO-VN on two-dimensional PAGE, as shown in Fig. 7. The result of chromatofocusing combined with immunodetection suggests that sialylation of vitronectin remained low with pI over pH 5 for the period from 24 to 72h after partial hepatectomy. The pI of PH-VN after 7d had recovered to pH 4.7–4.5 (data not shown).

Fig. 10. The elution pattern of vitronectin with descending elution pH. The amount of vitronectin was measured by the band intensity of an immunoblot of the eluted fraction and is expressed as %, when taking that of 1 μg of purified NO-VN as 100%. Solid line: PH-VN (24 h), chain line: NO-VN, dotted line: pH of the eluted fraction.

#### **3.1.4 Discussion**

92 Liver Regeneration

Healthcare Inc.) and a fast protein liquid chromatograph (FPLC; AKTA Purifier, GE Healthcare, Inc). The starting buffer was 0.025 M bis-Tris, pH 7.1, and the elution buffer was 10% Polybuffer74, pH 4.0. Buffers were filtered through 0.22 μm filters under vacuum and degassed. Samples were adjusted to the pH of the starting buffer, or exchanged by dialysis into the starting buffer. The column was pre-equilibrated with the starting buffer until the eluent was applied to the column, which was the same pH as that of starting buffer. After samples were applied to FPLC, the elution buffer was added. Samples were separated by the pI of each component protein in the column, and sequentially eluted from the column. The effluent was continuously monitored by absorbance at 280 nm and divided by 1 mL/tube. After the pH gradient ended, the column was washed with two column volumes of 2M NaCl to elute the molecules still bound to the column. Finally, the column was reequilibrated with five column volumes of starting buffer until UV absorbance and pH values reached a plateau. An aliquot (10 μL) of each fraction eluted from the column was subjected to SDS-PAGE on a 7% polyacrylamide gel, and the separated protein bands were electro transferred to a polyvinylidene difluoride membrane and detected with specific antibodies to vitronectin. Membranes were developed with ECL (GE Healthcare Inc.).

An example of the elution pattern of rat plasma is shown in Fig. 9. As the buffer flowed through the chromatofocusing column, the pH decreased and a descending pH gradient was generated. As shown in Fig. 10, when vitronectin in rat plasma was separated by chromatofocusing, NO-VN was eluted at pH 4.7–4.5, while PH–VN (24 h) was eluted at pH

Fig. 9. Chromatogram of non-operated rat plasma. Dotted line, pH; solid line, absorbance at 280 nm. Flow rate: 0.5 mL/min, temperature: 4°C. Other conditions are described in the

**3.1.3 Results** 

5.2-4.8.

text.

The elution pattern on chromatofocusing was very reproducible, and the pIs of NO-VN and PH-VN were found to be 4.7–4.5 and 5.2–4.8, respectively (Fig. 10). The immunostaining of PH-VN after two-dimensional PAGE (Fig. 7) indicated the presence of two components (pI 4.6 and 6.0), whereas PH-VN was eluted by chromatofocusing at intermediate pH, although the two methods agreed on the tendency for the pI of PH-VN to be considerably shifted toward alkalinity compared to that of NO-VN. This discrepancy may be due not only to the difference in the plasma sample lots but also because parts of the vitronectin eluted by chromatofocusing are multimerized in the buffer like the vitronectin in physiological plasma, which shows a broad pH range of elution peaks indicating variation in sialylation of the vitronectin molecules contained. Chromatofocusing has the advantage of analyzing the isoelectric property under physiological conditions, especially in detection of changes in sialylation of glycoproteins, and the eluted fractions are utilizable for activity measurements (Kneba, M., et al. 1983).

#### **3.1.5 Summary**

Each plasma sample was subjected to chromatofocusing using FPLC, which can separate molecules by their isoelectric point (pI). Fractions eluted from the column were subjected to

Matrix Restructuring During Liver Regeneration

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Gebb, C., Hayman, E.G., Engvall, E. & Ruoslahti, E. (1986) Interaction of vitronectin with

Hayman, E.G., Pierschbacher, M.D., Ohgren, Y. & Ruoslahti, E. (1983) Serum spreading

Holmes, R. (1967) Preparation from human serum of an alpha-one protein which induces the immediate growth of unadapted cells in vitro. *J Cell Biol,* 32, 297-308. Hughes, R.C. (1997) *Adhesive glycoproteins and receptors*. Elsevier Sciences B.V., New York. Ip, C. (1979) Effect of partial hepatectomy and hydrocortisone administration on liver and

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Key results: The chromatofocusing and immunoblotting of plasma before and after sialidase treatment enabled us to demonstrate when and what alteration of sialylation occurs in each glycoprotein at each different stage during liver regeneration. The changes in pI essentially coincided with that of 2D-PAGE; however, we can determine the pI of a sample under physiological condition by using the chromatofocusing technique. Furthermore, this method is facile, quick, and applicable to recovered samples for activity analyses because they are non-denatured and separated by pI.
