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Glycobiology Advance Access originally published online on April 27, 2005
Glycobiology 2005 15(9):827-837; doi:10.1093/glycob/cwi068
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© The Author 2005. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oupjournals.org

Inflammation-dependent changes in {alpha}2,3-, {alpha}2,6-, and {alpha}2,8-sialic acid glycotopes on serum glycoproteins in mice

Zenta Yasukawa2,3, Chihiro Sato2,3,4 and Ken Kitajima1,2,3,4

2 Laboratory of Animal Cell Function, Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan; 3 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; and 4 Institute for Advanced Research, Nagoya University, Nagoya 464-8601, Japan

1 To whom correspondence should be addressed; e-mail: kitajima{at}agr.nagoya-u.ac.jp

Received on September 28, 2004; revised on April 10, 2005; accepted on April 24, 2005


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
The expression of acute-phase serum proteins increases in response to inflammatory stimuli. Most of these proteins are glycoproteins that often contain sialic acids (Sia). It is unknown, however, how the expression of Sia in these glycoproteins changes during inflammation. This study demonstrates changes in the {alpha}2,3-, {alpha}2,6-, and {alpha}2,8-Sia glycotopes on serum glycoproteins in response to turpentine oil-induced inflammation, based on lectin- and immunoblot analyses by using sialyl linkage-specific lectins, Maackia amurensis for the {alpha}2,3-Sia glycotope and Sambucus sieboldiana for the {alpha}2,6-Sia glycotopes, and monoclonal antibody 2-4B (mAb.2-4B) recognizing the di- and oligomers of the {alpha}2,8-Neu5Gc residue. There was an increase in a limited number of sialoglycoproteins containing the {alpha}2,3-, {alpha}2,6-, or {alpha}2,8-Sia glycotopes. Reverse transcription–polymerase chain reaction (RT–PCR) analysis of the expression profiles of mRNAs for the known sialyltransferases in mouse liver during inflammation indicated the up-regulated expression of ß-galactoside {alpha}2,3-sialyltransferases (ST3Gal I and ST3Gal III) and ß-N-acetylgalactosaminide {alpha}2,6-sialyltransferase (ST6GalNAc VI) as well as ß-galactoside {alpha}2,6-sialyltransferase (ST6Gal I) mRNAs. Notably, ST3Gal I and III and ST6GalNAc VI are involved in the synthesis of the {alpha}2,3- and {alpha}2,6-Sia glycotopes on O-glycan chains and possibly on gangliosides, whereas ST6Gal I is specific for N-glycan chains. These results provide evidence for the inflammation-induced expression of sialyl glycotopes in serum glycoproteins. We demonstrated that inflammation significantly increased the expression of an unknown 32-kDa glycoprotein containing the {alpha}2,8-Sia glycotope. The mechanism for the increase in glycoprotein in inflamed mouse serum remains to be examined, as mRNA expression for all of the {alpha}2,8-sialyltransferases (ST8Sia I-VI) was unchanged during inflammation.

Key words: disialic acid / inflammation / oligosialic acid / serum glycoprotein / sialyltransferase


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
Sialic acid (Sia) residues often cap the terminal galactose residues of serum glycoproteins. The Sia residues regulate the lifetime of serum glycoproteins by prohibiting hepatic asialoglycoprotein receptor-dependent clearance of the galactose-exposed asialoglycoproteins from the blood stream (Ashwell and Morell, 1974Go). In some cases, Sia residues of lymphocyte glycoproteins regulate the activation state of lymphocytes through binding Sia-binding immunoglobulin superfamily lectins (siglecs) on the lymphocytes and other tissues (Crocker, 2002Go). To date, 11 siglecs have been identified in humans, and each siglec appears to have its own preference for the sialyl linkages (Crocker, 2002Go). The finding of a series of siglecs strengthened the idea that the temporal appearance and changes in the terminal sialyl linkages in serum glycoproteins are important for the regulation of glycoprotein functions. We recently demonstrated unique di- and oligosialic acid (diSia and oligoSia) structures in serum glycoproteins such as bovine fetuin, bovine {alpha}2-macroglobulin, and bovine adipoQ (Kitajima et al., 1999Go; Sato et al., 2001Go). Notably, these glycoproteins are not always modified by the {alpha}2,8-linked diSia residue in normal blood, and 1% of fetuin, 4% of {alpha}2-macroglobulin, and a small percentage of adipoQ molecules contain the diSia structures (Kitajima et al., 1999Go; Sato et al., 2001Go). These facts suggest that a small population of the sialoglycoproteins is specifically terminated by the {alpha}2,8-linked diSia under some physiologic conditions. Blood levels of {alpha}2-macroglobulin and adipoQ change in response to inflammatory stimuli and some hormones, respectively (Gehring et al., 1987Go; Hu et al., 1996Go). Thus, it is possible that the diSia structures have a role in inflammatory responses.

Inflammation is a local reaction of vascular epithelial cells in response to tissue injury (Baumann and Gauldie, 1994Go). Many physical and chemical factors such as hot/cold stimuli, wounds, lipopolysaccharides, turpentine oil, and tetrachloromethane cause inflammation. A major systemic event that occurs in mice following the induction of inflammation is a marked change in the levels of certain serum proteins, the so-called acute-phase proteins. Changes in the concentration of these serum proteins are regarded as sensitive indicators useful for diagnostic and prognostic assessments of inflammation and infection (Gabay and Kushner, 1999Go). Acute-phase proteins are induced by inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor-{alpha} (TNF-{alpha}), and IL-6 acting on liver tissue and triggering their biosynthesis and secretion (Gabay and Kushner, 1999Go).

Although most acute-phase proteins are glycoproteins, little attention has been paid to the alteration of glycotopes on glycoproteins during inflammation, except for concanavalin A (ConA)-reactive N-glycans (Heegaard, 1992Go). Structural changes of the N-linked glycans on some serum glycoproteins such as {alpha}1-acid glycoprotein, {alpha}1-esterase, and {alpha}1-protease inhibitor during inflammation were studied by using crossed affinity immunoelectrophoresis with ConA, demonstrating that the ConA glycotope on serum glycoproteins changes from ConA strongly bound forms to ConA weakly bound forms in mice (Heegaard, 1992Go). Therefore, we focused on changes in the expression of {alpha}2,3-, {alpha}2,6-, and {alpha}2,8-linked Sia glycotopes on serum glycoproteins, especially that of acute-phase glycoproteins under inflammatory conditions. In this study, we describe the linkage-specific changes of acute-phase glycoproteins using linkage-specific probes: Maackia amurensis lectin (MAM) for the 2,3-Sia glycotope, Sambucus sieboldiana lectin (SSA) for the {alpha}2,6-Sia glycotope, and monoclonal antibody 2-4B (mAb.2-4B) for the {alpha}2,8-Sia linkage. We also describe the inflammation-induced changes in the expression of mRNAs for the sialyltransferases that have been cloned so far: ST6Gal I–II, ST3Gal I–V, ST6GalNAc I–VI, and ST8Sia I–VI.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Acknowledgments
 References
 
Changes of the expression of ConA glycotopes in serum glycoproteins of inflamed mice
Mice were injected subcutaneously with turpentine oil, and their sera were collected before and 2 and 4 days after the injection. The collected sera were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)/Coomassie Brilliant Blue staining to examine the expression level of the serum glycoproteins. The expression level of the proteins at 124, 68, 45, 37, and 32 kDa increased after the inflammatory stimulus (Figure 1a). Based on the molecular masses of the major acute-phase proteins in mice (Baumann et al., 1983Go), the 68-kDa protein corresponded to hemopexin and 45-kDa proteins corresponded to haptoglobin ß chain and {alpha}1-acid glycoprotein. The increase in the haptoglobin ß chain was confirmed by western blotting (Figure 1b).



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  		Fig. 1. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)/Coomassie Brilliant Blue stain and the concanavalin A (ConA) lectin blotting of whole serum in mice before and after turpentine oil-induced inflammation. Three mice (No. 1–3) were subcutaneously injected with turpentine oil at day 0. Before (day 0) and 2 and 4 days after the injection (day 2 and day 4), blood was collected and sera were prepared. Half a microliter of sera (15 µg as protein) was subjected to SDS–PAGE (upper gel, 3%; lower gel, 10% polyacrylamide gel). (a) The gel was stained with Coomassie Brilliant Blue. The arrows indicate molecular masses. Positions of molecular mass markers are indicated on the left. Acute-phase proteins are indicated by the arrows and the molecular masses on the right of the panel. (b) After SDS–PAGE/electroblotting on the polyvinylidene difluoride (PVDF) membrane, the membrane was immunostained by using anti-haptoglobin ß chain antibody as a primary antibody. The haptoglobin ß chain was detected at 45 kDa. (c) After SDS–PAGE/electroblotting on the PVDF membrane, the membrane was subjected to the lectin staining by using biotinylated ConA (biotin-ConA) and the avidin–biotin-peroxidase complex system. No stain was observed without biotin-ConA (data not shown).

 

The SDS–PAGE profiles of ConA-reactive glycoproteins in mouse serum before and after the inflammatory stimulus are shown in Figure 1c. Many ConA-reactive glycoproteins were detected in mouse sera. There were no obvious changes in their expression, except for the 45-kDa and 32-kDa glycoproteins. The expression of these ConA-reactive glycoproteins increased 2 and 4 days after inducing the inflammation. The 45-kDa glycoprotein was identified as haptoglobin ß chain and {alpha}1-acid glycoprotein, based on a previous report (Mackiewicz et al., 1991Go).

Changes in serum sialoglycoprotein expression in inflamed mice
SSA and MAM glycotopes. To examine changes in the suppression of monosialic acid residue-containing glycoproteins, the sialyl linkage-specific lectins, MAM (specific for Sia{alpha}2Æ3Galß1Æ4GlcNAc or glucose) and SSA (specific for Sia{alpha}2Æ6Gal or GalNAc), were used for the lectin-blotting analyses. The amount of two MAM-reactive glycoproteins (68 and 45 kDa) and that of four SSA-reactive glycoproteins (115, 68, 45, and 32 kDa) were increased 2 and 4 days after the inflammation stimulus (Figure 2a and b). It is reported that MAM binds not only Sia{alpha}2->3Galß1Æ4GlcNAc but also SO3->3Galß1Æ4GlcNAc (Bai et al., 2001Go). However, the MAM glycotopes in Figure 2a were sialylated ones because they were all sensitive to sialidase digestion (data not shown). The results of densitometric quantification of each component are shown in Figure 2c–f. The amount of SSA glycotope on 115-kDa glycoprotein increased 2.2-fold 2 days after stimulation (day 2) and decreased at day 4 (Figure 2c). The amount of SSA glycotope on 68-kDa glycoprotein increased by 2.0-fold until day 4, whereas that of MAM glycotope increased by 2.0-fold at day 2 and remained unchanged until day 4 (Figure 2d). The amount of SSA and MAM glycotopes on 45-kDa components increased 20-fold and 8.6-fold, respectively, at day 2 (Figure 2e). The amount of SSA glycotope on 32-kDa glycoprotein increased two-fold by day 4 (Figure 2f).



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  		Fig. 2. The MAM and SSA lectin blotting of whole serum in mice before and after turpentine oil-induced inflammation for nonreducing terminal {alpha}2,3- and {alpha}2,6-sialic acid glycotopes. The same serum samples as shown in Figure 1 were analyzed. See also the legend of Figure 1. The whole sera (0.5 µL/lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% polyacrylamide gel and blotted on the membrane for the lectin blotting as described under Materials and methods. (a) MAM, which is specific to Sia{alpha}2Æ3Galß1Æ4GlcNAc or Glc structure, and (b) SSA, which is specific to Sia{alpha}2Æ6Gal or GalNAc structure. Control incubation without the lectins was carried out (data not shown), and the nonspecifically stained bands are indicated by shown), and the nonspecifically stained bands are indicated by the asterisks.. (c–d) Expression profiles of the glycotope amount in (c) 115-kDa, (d) 68-kDa, (e) 45-kDa, and (f) 32-kDa glycoproteins. The glycotope amount was densitometrically quantified by using an ATTO Lane & Spot Analyzer (ATTO, Tokyo, Japan). The amount of MAM (square) or SSA glycotope (circle) at each day represents the relative value, and the average value at day 0 is set equal to 1.0. The standard deviation is indicated by the bar (n = 3). (g) A proportion of the amount of MAM (square) and SSA (circle) glycotope on hemopexin to the protein amount of hemopexin. The protein amount of hemopexin (Figure 1a) was densitometrically determined. The average value at day 0 is set equal to 1.0. The standard deviation is indicated by the bar (n = 3).

 

Of those glycoproteins, the 68-kDa glycoprotein was determined to be hemopexin by the peptide mass mapping using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of trypsin digests. Hemopexin is an acute-phase glycoprotein (Baumann et al., 1983Go) and reported to be MAM positive (Yamamoto et al., 1998Go). The 45-kDa glycoproteins were haptoglobin ß chain and {alpha}1-acid glycoprotein, because these glycoproteins have both MAM and SSA glycotopes (Pousset et al., 1997Go; Yamamoto et al., 1998Go; Roemer et al., 2001Go). To determine whether the level of SSA and MAM glycotopes increased on these glycoproteins or whether the level of glycoproteins containing these glycotopes increased, the amount of SSA or MAM glycotope on hemopexin (Figure 2a and b) to the protein amount of hemopexin (Figure 1a) is calculated based on the densitometric analyses (Figure 2g). The proportions remained unchanged before and after the inflammation stimulus, suggesting that the amount of these glycotopes on hemopexin does not change. It cannot be concluded that this is also the case with the 45-kDa acute-phase glycoproteins, {alpha}1-acid glycoprotein, and haptoglobin, because these glycoproteins could not be individually quantified because of a shortage of presently available antibodies applicable for immunoblotting or immunoprecipitation.

Di- and oligoSia glycotopes

We also analyzed changes in the expression of the di- and oligoSia-containing glycoproteins in mouse sera before and after the inflammatory stimulus. The results of western blotting of the individual sera collected from three inflamed mice using mAb.2-4B, which specifically recognizes Neu5Gc{alpha}2Æ(8Neu5Gc{alpha}2->)n–1, n ≥ 2, as a primary antibody, are shown in Figure 3. Fewer glycoproteins were detected with mAb.2-4B as compared with those detected by lectin-blotting using MAM and SSA (Figure 2a and b). In mouse sera, the 120-kDa, 70-kDa, 32-kDa, and 30-kDa glycoproteins reacted to mAb.2-4B. After the inflammatory stimulus, levels of mAb.2-4B glycotope at 32 kDa increased in all three mice (Figure 3), whereas those levels before the stimulus were different from mouse to mouse: no expression in mouse 1 and a slight expression in mice 2 and 3. These results suggest two possibilities. One is that the mAb.2-4B glycotope increases on the preexisting 32-kDa glycoprotein after the stimulus, and another is that a 32-kDa glycoprotein containing the mAb.2-4B glycotope is newly expressed. We cannot conclude which is correct until the 32-kDa glycoprotein is identified. On the other hand, the mAb.2-4B glycotope on the 120-kDa, 70-kDa and 30-kDa glycoproteins did not change or decrease after the inflammatory stimulus. These results indicate that the 32-kDa glycoprotein is not only a new member of the di- and oligoSia-containing glycoprotein family, but also an acute-phase protein. Furthermore, the mAb.2-4B glycotope on the 32-kDa glycoprotein disappeared when this glycoprotein was blotted onto polyvinylidene difluoride (PVDF) membranes treated with mild alkaline solution, suggesting that di- and oligoNeu5Gc residues are present on O-linked glycan, but not on N-linked glycan chains. Western blot analyses using mAb.OL28, which recognizes (Neu5Ac)n, n ≥ 4, and mAb.S2-566, which recognizes Neu5Ac{alpha}2->8Neu5Ac{alpha}2->3Gal, as primary antibodies had profiles similar to those of mAb.2-4B (Figure 3), except for the 70-kDa glycoprotein (data not shown). The 70-kDa glycoprotein was not recognized by mAb.OL28 or mAb.S2-566. These results indicate that the 120-kDa, 32-kDa, and 30-kDa glycoproteins contain not only di- and oligoNeu5Gc, but also di- and oligoNeu5Ac, and that the 70-kDa glycoprotein exclusively bears the Neu5Gc{alpha}2->8Neu5Gc structure.



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  		Fig. 3. The monoclonal antibody 2-4B (mAb.2-4B) immunostaining of whole serum in mice before and after turpentine oil-induced inflammation for the {alpha}2,8-sialic acid glycotope. The same serum samples as shown in Figure 1 were analyzed. See also the legend of Figure 1. The whole sera (0.5 µL/lane) were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% polyacrylamide gel, and blotted on the membrane for the immunostaining with mAb.2-4B [specific to NeuGc{alpha}2->(8NeuGc{alpha}2->)n–1, (n ≥ 2) structure] as described under Materials and methods. Control incubation without the primary antibody was carried out (data not shown), and the nonspecifically stained bands are indicated by ) A proportion of the amount of MAM (square) and SSA (circle) glycotope on hemopexin to the protein amount of hemopexin. The protein amount of hemopexin (Figure 1a) was densitometrically determined. The average value at day 0 is set equal to 1.0. The standard deviation is indicated by the bar (n = 3)..

 

Changes in the mRNA expression level for various glycosyltransferases
Injection of turpentine oil into mice induces an increase in the expression level of the {alpha}2,3- and {alpha}2,6-Sia glycotopes on several glycoproteins and in the expression of the {alpha}2,8-Sia glycotope on 32-kDa glycoprotein in serum (Figures 2 and 3). Therefore, we examined whether the increase in these glycotopes is due to inflammation-induced changes in the expression of the related sialyltransferases. The liver is the main organ that synthesizes serum acute-phase proteins (Baumann and Gauldie, 1994Go). To determine which sialyltransferases are induced in the liver by an inflammatory stimulus, we performed reverse transcription–polymerase chain reaction (RT–PCR) analyses for mRNAs of five ß-galactoside {alpha}2,3-sialyltransferases (ST3Gal I, II, III, IV, and V), six ß-N-acetylgalactosaminide {alpha}2,6-sialyltransferases (ST6GalNAc I, II, III, IV, V, and VI), two ß-galactoside {alpha}2,6-sialyltransferases (ST6Gal I and II), and six {alpha}-sialoside {alpha}2,8-sialyltransferases (ST8Sia I, II, III, IV, V, and VI). Livers were excised from mice before (day 0) and 1, 2, 3, and 4 days after the inflammatory stimulus (days 1–4), and total RNA was extracted and used for RT–PCR. The results are shown in Figures 4–Go6 and summarized in Table I.



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  		Fig. 4. Expression of the mRNAs for ST6Gal I and haptoglobin in mouse liver before and after turpentine oil-induced inflammation as analyzed by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). (a) RT–PCR products were analyzed on 1% agarose electrophoresis as described under Materials and methods. Three mice were killed before (day 0) and 1, 2, 3, and 4 days after the inflammation stimulus (day 1–4) to prepare the template cDNAs from the livers. The primers are described in Table II. Amplification cycle numbers are 32 for ST6Gal I, 26 for haptoglobin, and 25 for GAPDH, where the amplification reactions linearly proceed. Expression profiles of the mRNAs for (b) ST6Gal I and (c) haptoglobin. The values at each day represent relative mRNA amounts of ST6Gal I and haptoglobin to that of glyceraldehyde-3-phosphate dehydrogenase mRNA, and the average values at day 0 are set equal to 1.0. The standard deviations are indicated by the bar (n = 3).

 


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  		Fig. 5. Expression profiles of the mRNAs for (a) ST3Gal I, (b) ST3Gal III, and (c) ST6GalNAc VI in mouse liver before and after turpentine oil-induced inflammation as analyzed by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). RT–PCR was carried out by using the same template cDNAs as in Figure 4. The primers are summarized in Table II. Amplification cycle numbers are 32 for ST3Gal I, 35 for ST3Gal III, 34 for ST6GalNAc VI, and 25 for glyceraldehyde-3-phosphate dehydrogenase, where the amplification reactions linearly proceed. See also the legend of Figure 4.

 


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  		Fig. 6. Expression profiles of the mRNAs for ST8Sia III and ST8Sia VI in mouse liver before and after turpentine oil-induced inflammation as analyzed by semiquantitative reverse transcription–polymerase chain reaction (RT–PCR). (a) RT–PCR products were analyzed by 1% agarose electrophoresis/blotting on Hybond-N+ membrane by using the digoxigenin (DIG)-labeled DNA probes as described under Materials and methods. RT–PCR was performed by using the same template cDNAs as in Figure 4. The primers are described in Table II. Amplification cycle numbers are 35 for ST8Sia III and VI and 25 for glyceraldehyde-3-phosphate dehydrogenase and under the cycle numbers, the amount of amplified DNA did not reach a plateau. Expression profiles of the mRNAs for ST8Sia III (b) and ST8Sia VI (c). See also the legend of Figure 4.

 

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  		Table I. Summary of the expression changes of the mRNAs for sialyltransferases in mouse liver before and after turpentine oil-induced inflammation

 


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  		Table II. Primers for reverse transcription–polymerase chain reaction (RT–PCR) analyses

 

Haptoglobin and ST6Gal I
Induction of mRNA expression for the known acute-phase proteins, ß-galactoside {alpha}2,6-sialyltransferase (ST6Gal I) and haptoglobin, was analyzed. Inflammation increased ST6Gal I and haptoglobin mRNAs by three- to four-fold and 7- to 12-fold, respectively (Figure 4a–c). These results coincide with previous results (Pajovic et al., 1994Go; Dalziel et al., 1999Go). The expression of ST6Gal II mRNA could not be detected (data not shown).

ST3Gal family
Of the ST3Gal family, the expression of the ST3Gal I and III mRNAs was changed before (day 0) and after the inflammatory stimulus (days 2–4). ST3Gal I expression increased by three-fold at day 1 compared with that at day 0 and thereafter decreased to the baseline level (Figure 5a). The ST3Gal III expression gradually increased until day 3 to reach a three-fold higher level compared with that at day 0 and decreased at day 4 (Figure 5b). Because the expression level of ST3Gal II mRNA in the liver was much lower than that of the rest of the ST3Gal family, it was probed by the Southern blot analysis. ST3Gal II expression was not detected before the inflammatory stimulus but, in some mice, detected after the inflammatory stimulus (data not shown). ST3Gal IV and V expression was detected in liver, and the level of expression remained unchanged (data not shown).

ST6GalNAc family
In the ST6GalNAc family, which is responsible for the synthesis of the Sia{alpha}2->6GalNAc linkage, changes were observed in the expression of mRNA for ST6GalNAc VI. ST6GalNAc VI expression gradually increased up to day 3 after the inflammation stimulus and decreased at day 4 (Figure 5c). The maximum expression level was three times higher than that at day 0. ST6GalNAc I and II mRNAs were constitutively expressed in liver (data not shown). The expression of mRNAs for ST6GalNAc III, IV, and V could not be detected in these mice (data not shown).

ST8Sia family
In the ST8Sia family, the expression of ST8Sia III, IV, V, and VI mRNAs was detected in liver. There was no effect of inflammation on the expression of the mRNAs for any of the ST8Sia family members. The expression levels for ST8Sia III, which catalyzes the transfer of a Sia residue onto the Sia{alpha}2->3Galß1->4GlcNAc structure on glycoproteins and glycolipids, and for ST8Sia VI, which is considered to be involved in the sialylation of monosialylated O-glycans preferentially (Takashima et al., 2002Go), are shown in Figure 6a–c. Both mRNAs were constitutively expressed in mouse liver, although the expression levels differed from mouse to mouse. The mRNAs for ST8Sia IV (an {alpha}2,8-polysialyltransferase) (Angata and Fukuda, 2003Go) and ST8Sia V (a sialyltransferase-preferring gangliosides) (Kono et al., 1996Go) were also expressed constitutively, like ST8Sia III and VI (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Acknowledgments
 References
 
From lectin blotting with SSA (specific for Neu5Ac or Neu5Gc{alpha}2->6Gal or GalNAc) and MAM (specific for Neu5Ac or Neu5Gc{alpha}2->3Galß1->GlcNAc or Glc) and the immunostaining with mAb.2-4B (specific for ({alpha}2->8Neu5Gc)n, n ≥ 2), we demonstrated the expression of particular serum glycoproteins containing the {alpha}2,3-, {alpha}2,6- and, {alpha}2,8-linked Sia residues changed by turpentine oil-induced inflammation. As expected, the {alpha}2,3- and {alpha}2,6-Sia residues are the prevalent glycotopes on serum glycoproteins (Figure 2). The expression level of only a limited number of glycoproteins bearing {alpha}2,3- and {alpha}2,6-Sia glycotopes increased, that is, the 68-kDa and 45-kDa glycoproteins for MAM and the 115-kDa, 68-kDa, 45-kDa, and 32-kDa glycoproteins for SSA. Although the inflammation-induced increase in the {alpha}2,3-Sia glycotope on the 68-kDa and 45-kDa glycoproteins is novel, the increased expression of the {alpha}2,6-Sia glycotope is widely acknowledged (Appenheimer et al., 2003Go). The 68-kDa glycoprotein was determined to be hemopexin, which is an acute-phase glycoprotein (Baumann et al., 1983Go), and reported to be recognized by MAM (Yamamoto et al., 1998Go). The 45-kDa glycoproteins were determined to be haptoglobin ß chain and {alpha}1-acid glycoprotein, because these glycoproteins are acute-phase glycoproteins and contain both {alpha}2,3- and {alpha}2,6-Sia glycotopes at their nonreducing ends of N-glycans (Pousset et al., 1997Go; Yamamoto et al., 1998Go; Roemer et al., 2001Go). It is difficult to conclude whether the concomitant increase of the {alpha}2,3- and {alpha}2,6-Sia glycotopes on these glycoproteins during inflammation is due to the increased expression of these glycoproteins or due to enhanced {alpha}2,3- or {alpha}2,6-sialylation on these glycoproteins. However, at least for hemopexin, the apparent increase of the {alpha}2,3- and {alpha}2,6-Sia glycotopes results from the increased expression of the glycoprotein because the proportion of the amounts of these glycotopes to the protein amount remains constant before and after the inflammation stimulation.

In contrast with the prevalence of the {alpha}2,3- and {alpha}2,6-Sia glycotopes, the {alpha}2,8-Sia glycotope is restricted to four glycoproteins of 120, 70, 32, and 30 kDa in mouse sera. Of those, only the 32-kDa glycoprotein is an inflammation-induced component. The finding that inflammation increases the {alpha}2,8-Sia glycotope on the serum glycoprotein is novel.

To gain insight into the mechanisms for the increase in the {alpha}2,3- and {alpha}2,8-linked Sia residues in certain serum glycoproteins of the turpentine oil-induced inflamed mice, we investigated the expression of the known cloned sialyltransferases in mouse liver before and after the inflammatory stimulus. Of those sialyltransferases, the expression of mRNA of ST3Gal I, ST3Gal III, and ST6GalNAc VI, as well as ST6Gal I, is up-regulated during inflammation. ST6Gal I is the only sialyltransferase whose expression is up-regulated in the liver during inflammation (Wang et al., 1989Go; Dalziel et al., 1999Go). The up-regulated expression of ST6Gal I has been discussed in relation to the increased expression of the {alpha}2,6-Sia glycotope during inflammation (Appenheimer et al., 2003Go).

With respect to the inflammation-induced increase of the {alpha}2,3-Sia glycotope in the mouse serum glycoproteins, this study revealed that the expression of mRNAs for ST3Gal I and III increases in liver during inflammation. It has been demonstrated that ST3Gal I is specific for Galß1->3GalNAc on O-glycans, and ST3Gal III prefers the type 1 N-acetyllactosamine (Galß1->3GlcNAc) (Kono et al., 1997Go) and possibly sialylates both N- and O-glycans. Thus, the elevated expression of these enzymes might be involved in the increased expression of the {alpha}2,3-Sia glycotopes in the serum glycoproteins during inflammation. Interestingly, an elevated expression of mRNAs for ST3Gal I and III was also demonstrated in human tracheal gland cells and bronchial mucosa in TNF-{alpha}-stimulated inflammation (Delmotte et al., 2001Go, 2002Go). Therefore, ST3Gal I and III appear to be involved in the increase of the {alpha}2,3-Sia glycotope on glycoproteins upon inflammation. The {alpha}2,6-, but not {alpha}2,3-, sialyltransferase activity increases in liver after inflammation (Kaplan et al., 1983Go; Lammers and Jamieson, 1986Go). In these studies, however, Galß1->4Glc was used as a substrate for the sialyltransferase activity assay, and the failure to detect the {alpha}2,3-sialyltransferase activity in the liver of inflamed mice might be due to the substrate specificity of ST3Gal I and III.

We demonstrated that inflammation increases the expression of ST6GalNAc VI mRNA. The expression of the ST6GalNAc VI mRNA is ubiquitous in various mouse organs, including liver (Okajima et al., 2000Go). ST6GalNAc VI catalyzes the transfer of Neu5Ac to the 6-O-position of a GalNAc residue in the Neu5Ac{alpha}2->3Galß1->3GalNAc structure. ST6GalNAc VI prefers gangliosides as substrates and best sialylates GM1b to produce GD1a (Okajima et al., 2000Go). Notably, bovine fetuin can be a substrate of this enzyme, because fetuin contains the Neu5Ac{alpha}2->3Galß1->3GalNAc structure on the O-glycans (Okajima et al., 2000Go). Therefore, ST6GalNAc VI might be involved in the increased formation of {alpha}2,6-sialylated O-linked glycans, that is, Neu5Ac{alpha}2->3Galß1->3(Neu5Ac2->6)GalNAc. It would also be of interest to determine whether the {alpha}2,6-Sia glycotope level increases on gangliosides in the liver of inflamed mice.

The elevation of the liver ST6Gal I mRNA level during inflammation is mediated by the inducible, liver-specific promoter-regulatory region, P1 (Dalziel et al., 1999Go). This region contains a CCAAT/enhancer binding proteins (C/EBP) consensus region at position –89 (Akira, 1997Go) and might be involved in the acute-phase reaction (Hu et al., 1997Go). In this regard, there was a C/EBP consensus region at position –378 in the 5' flanking region of ST3Gal III gene (NM_009176 [GenBank] ), and this region might be involved in the acute-phase reaction to induce the expression of ST3Gal III. For ST3Gal I and ST6GalNAc VI genes, other promoter–regulatory regions might be operative in the acute-phase reaction, because to our knowledge there is no C/EBP consensus sequence.

Of all the ST8Sia mRNAs, those for ST8Sia III, IV, V, and VI are expressed in liver. As far as we could determine, however, the expression of all ST8Sia mRNAs is constitutive and remains unchanged by inflammation. Therefore, the expression of the 32-kDa glycoprotein containing the {alpha}2,8-Sia glycotope is up-regulated (Figure 3). We do not know why this happens, but we can speculate the mechanism if the {alpha}2,8-Sia glycotope is synthesized in liver like other acute-phase proteins. The {alpha}2,8-Sia glycotope is suggested to be on the O-glycan of the 32-kDa glycoprotein (see Results). It is interesting that the expression of mRNAs for ST3Gal I, ST3Gal III, and ST6GalNAc VI is up-regulated because all these enzymes use O-glycans as the substrate, as discussed above. For example, once the synthesis of the Sia{alpha}2->3Gal structure by ST3Gal I or ST3Gal III or the synthesis of the Sia{alpha}2->6GalNAc structure by ST6GalNAc VI is enhanced on the O-glycan at inflammation, the subsequent addition of an {alpha}2,8-Sia residue on the Sia residues might occur voluntarily by the constitutively expressed ST8Sia III and/or ST8Sia VI, both of which are involved in the formation of the Sia{alpha}2->8Sia structure on O-glycan chains (Sato et al., 2001Go; Takashima et al., 2003Go). Further studies are necessary, however, to identify the 32-kDa glycoprotein as well as to determine cell types expressing the glycoprotein. Further characterization of the 32-kDa glycoprotein is under way in our laboratory.

The ubiquitous presence of {alpha}2,8-linked di- and oligoSia glycotopes was recently established by our group (Sato, 2004Go). In bovine serum glycoproteins, fetuin and {alpha}2-macroglobulin secreted from liver (Kitajima et al., 1999Go) and adipoQ secreted from adipose tissues (Sato et al., 2001Go) contain this structural unit. In other sources, the {alpha}5 subunit of integrin from human melanoma (Nadanaka et al., 2001Go), CD166 from brain (Sato et al., 2002Go), and T lymphocytes (Sato, 2004Go) and CD36 from human milk (Yabe et al., 2003Go) also contain this glycotope. Interestingly, this structural unit on CD166 is involved in neurite formation in Neuro2A cells (Sato et al., 2002Go). The biologic significance of other di- and oligoSia-containing glycoproteins, however, remains unknown. Because we demonstrated that the 32-kDa glycoprotein containing the {alpha}2,8-Sia glycotope is increased in sera of inflamed mice, current efforts are aimed at revealing how the {alpha}2,8-Sia glycotope on the 32-kDa glycoprotein is involved in inflammation. In this regard, it is important to search for a binding counterpart of the {alpha}2,8-Sia glycotope in the 32-kDa glycoprotein. The {alpha}2,6-linked Sia residues on haptoglobin ß chain are recognized by siglec-2 (CD22), and their possible involvement in B-cell activation in acquired immunity has been discussed (Hanasaki et al., 1995Go). Likewise, it is reported that siglec-1, -5, -7, and -11 have the ability to bind the {alpha}2,8-Sia linkages at least in vitro (Crocker, 2002Go, 2004Go). Furthermore, siglec-1 is highly expressed on inflammatory macrophages and is suggested to mediate cell–cell and/or cell–substrate interactions in a range of pathologic conditions, as well as under normal conditions (Crocker et al., 1997Go). It would be thus interesting to determine whether the 32-kDa glycoprotein is a ligand for siglec-1 or for any of the other siglecs.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Acknowledgments
 References
 
Materials
Enhanced chemiluminescence (ECL) reagents and protein G-Sepharose resins were purchased from Amersham Biosciences (Piscataway, NJ). PVDF membrane (Immobilon P) was a product of Millipore (Bedford, MA). Molecular marker was purchased from Sigma (St. Louis, MO) and BIO-RAD (Hercules, CA). Peroxidase-conjugated rat anti-mouse IgG was purchased from American Qulex (San Clemente, CA). Peroxidase-conjugated rat anti-mouse IgM was purchased from Zymed Laboratories (San Francisco, CA). Mouse monoclonal IgM antibody 2–4B, which recognizes Neu5Gc{alpha}2->(8Neu5Gc{alpha}2->)n–1, n ≥ 2, was prepared as described previously (Sato et al., 1998Go). Peroxidase-conjugated rabbit anti-goat IgG, goat antiserum to human haptoglobin ß chain, and rat anti-mouse IgM were purchased from Cappel (West Chester, PA). Maackia amurensis agglutinin lectin (MAM), which recognizes Sia{alpha}2->3Galß1->4GlcNAc or Glc, and Sambucas sieboldiana agglutinin lectin (SSA), which recognizes Sia{alpha}2->6Gal or GalNAc, were purchased from Seikagaku Co. (Tokyo, Japan). Mouse anti-lectin antiserum to MAM and SSA was kindly provided by Dr. Tsukasa Matsuda (Nagoya University, Nagoya, Japan). Biotinylated ConA (biotin-ConA) was purchased from Honen (Tokyo, Japan). Peroxidase-conjugated anti-digoxigenin (DIG) Fab fragments were purchased from Roche (Mannheim, Germany). Vectastain ABC kit was purchased from Vector Laboratories (Burlingame, CA). Male, 8-week-old ddY mice were obtained from Japan SLC (Hamamatsu, Japan). Turpentine oil was kindly provided by Dr. Hiroaki Oda (Nagoya University, Nagoya, Japan).

Experimental inflammation
Mice were injected subcutaneously with 240 µL of turpentine oil. Blood was collected before and 2 and 4 days after the inflammation stimulus. Blood was stored at 25°C for 1 h and subsequently at 4°C overnight. After centrifugation at 1000 x g for 5 min, the supernatants were used.

SDS–PAGE, western blotting, and lectin blotting
Samples were dissolved in Laemmli buffer with 5% mercaptoethanol and boiled at 100°C for 3 min. The samples were electrophoresed on 10% polyacrylamide gels and visualized by Coomassie Brilliant Blue or electroblotted onto PVDF membranes by using a semidry blotting apparatus, as described by Sato et al. (2000)Go. The membrane was blocked with 10 mM sodium phosphate buffer (pH 7.2) and 0.15 M NaCl (PBS) with 0.05% Tween 20 (PBS-T) containing 1% bovine serum albumin (BSA) at 25°C for 1 h, as described by Sato et al. (2000)Go. For western blotting, the membrane was incubated with a primary antibody, mAb.2-4B (0.50 µg/mL), or goat antiserum to human haptoglobin ß chain (5.0 µg/mL) at 4°C for 16 h. For secondary antibody, peroxidase-conjugated anti-mouse IgM (1/5000 diluted) or anti-goat IgG (1/3000 diluted) was used. For the lectin blotting using MAM or SSA, the membrane was incubated with MAM (1.0 µg/mL) or SSA (1.0 µg/mL) at 4°C for 16 h. The membrane was washed with PBS-T and incubated with mouse anti-lectin antiserum to MAM (1/1000 diluted) or to SSA (1/1000 diluted) at 37°C for 2 h. The membrane was washed with PBS-T and incubated with peroxidase-conjugated anti-mouse IgG (0.1 µg/mL) at 37°C for 45 min. The color development was carried out as described by Sato et al. (2000)Go. For lectin blotting using biotin-ConA, the membrane was incubated with biotin-ConA (1.0 µg/mL) at 4°C for 16 h. After washing with PBS-T, visualization was carried out by using Vectastain ABC kit.

Peptide mass mapping using MALDI-TOF MS
After SDS–PAGE/Coomassie Brilliant Blue staining, a stained gel was excised, cut into pieces, put into siliconized tubes, and destained. The gel pieces were incubated in 100 µL of 10 mM dithiothreitol, 100 mM NH4HCO3 (pH 8.7), and 0.3% ethylenediaminetetraacetic acid (EDTA) for 15 min and then in 100 µL of 100 mM acrylamide, 100 mM NH4HCO3 (pH 8.7), and 0.3% EDTA for 15 min. After washing four times with 500 µL of 10% acetic acid/methanol (1:1, v/v), the gel pieces were equilibrated in 50 µL of 100 mM NH4HCO3 (pH 7.9), dehydrated by adding 100 µL of 100% CH3CN, and dried by Speed Vac (Savant Instrument, Farmingdale, NY). To this, 25 µL of trypsin (0.05 µg, Promega, Madison, WI) dissolved in 100 µM NH4HCO3 (pH 7.9) was added, and the mixture was digested at 37°C for 12 h. The peptides were extracted with 50 µL of 0.1% trifluoroacetic acid/acetonitrile (1:1 and 1:2, v/v), successively, and analyzed by MALDI-TOF MS using 4700 Proteomics Analyser (Applied Biosystems, Framingham, MA) operated in the MS mode to generate peptide mass fingerprinting. {alpha}-Ciano-4-hydroxycinnamic acid (Sigma) was used as a matrix. The mass spectra obtained were referenced to the MSDB database using the Mascot search engine (http://www.matrixscience.com/search_form_select.html).

RT–PCR
Total RNA was prepared from mouse liver by using Trizol (Life Technologies, Tokyo, Japan) according to the manufacturer’s instructions. Total RNA (2 µg) was reverse transcribed by using 200 units of SUPERSCRIPT II (Invitrogen, Carlsbad, CA), 50 ng of random hexamer (GIBCO, Rockville, MD), and 10 mM dNTPs in buffer supplied by the manufacturer and a reaction volume of 20 µL at room temperature for 10 min and at 42°C for 50 min. The reaction was stopped at 94°C for 5 min. The cDNA synthesized from the total RNA was amplified for 25–35 cycles in a 15-µL reaction mixture containing sense and antisense primers (200 pmol each), 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, all four dNTPs (each 200 µM), and 0.375 unit of Taq polymerase (Takara) on a thermal cycler. Each cycle involved incubation at 94°C for 1 min, at 54 or 55°C for 1 min, and at 72°C for 2 min. The oligonucleotide primers used in this study are summarized in Table II. The PCR amplification was found to be proportional to the initial amount of the cDNAs with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the number of cycles (20–25 cycles) of PCR (data not shown). Aliquots of the PCR products were separated on a 1.0% agarose gel containing 0.5 µg/mL of ethidium bromide and visualized by exposing ultraviolet (UV) or blotted onto Hybond-N+ membranes (Amersham Biosciences). The membranes were then probed with the DIG-labeled DNA that had been amplified from mouse adult brain cDNA (except for ST6GalNAc I that had been amplified from mouse adult mammary gland cDNA) by using PCR and labeled by DIG using DIG DNA Labeling Kit (Roche). The membranes were visualized as described by Sato et al. (2000)Go.


   Acknowledgments
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
References
 
We thank Dr. Tsukasa Matsuda (Nagoya University, Nagoya, Japan) for his valuable discussion. This research was supported in part by CREST of Japan Science and Technology Corporation (to K.K.), the 21st Century COE Program (to K.K.), Young Scientists (B) (16770073) (to C.S.), and the Japan Society for the Promotion of Science Research Fellows (16005841) (to Z.Y.) from the Ministry of Education, Science, Sports and Culture, and Mizutani Foundation (to C.S.).


   Abbreviations
 
Biotin-ConA, biotinylated ConA; ConA, concanavalin A; DIG, digoxigenin; diSia, disialic acid; mAb, monoclonal antibody; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; oligoSia, oligosialic acid; PBS, phosphate-buffered saline; PBS-T, PBS containing 0.05% Tween 20; PVDF, polyvinylidene difluoride; RT–PCR, reverse transcription–polymerase chain reaction; Sia, sialic acid; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; ST3Gal, {alpha}2,3-sialyltransferase; ST6Gal/GalNAc, {alpha}2,6-sialyltransferase; ST8Sia, {alpha}2,8-sialyltransferase.


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Acknowledgments
 References
 
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