2 Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; 3 CREST, JST Kawaguchi 332-1102, Japan; 4 RIKEN (Institute of Physical and Chemical Research), Saitama 351-0198, Japan; 5 Third Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; 6 Department of Molecular Pathology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
Received on October 20, 2004; revised on November 1, 2004; accepted on November 2, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: C-mannosylation / diabetes / hyperglycemia / mannosyltransferase / thrombospondin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
C-mannosylation is a novel type of protein glycosylation discovered by Hofsteenge et al. (1994). C-mannosylation is unique in that an
-mannose is directly bound to the indole C2 carbon atom of a Trp residue through a C-C bond to produce C-mannosylated Trp (CMW) (Figure 1). C-mannosylation occurs at the first Trp in the consensus amino acid sequence Trp-X-X-Trp in proteins. There are several examples of C-mannosylated proteins, including RNase2, interleukin-12, complements (C6, C7, C8a, C8b, and C9), properdin, thrombospondin, F-spondin, the erythropoietin receptor, and mucins (MUC5AC and MUC5B) (De Peredo et al., 2002
; Furmanek and Hofsteenge, 2000
; Furmanek et al., 2003
; Hartmann and Hofsteenge, 2000
; Hofsteenge et al., 2001
; Perez-Vilar et al., 2004
). C-mannosylation is thought to be synthesized by a specific unidentified mannosyltransferase localized in the microsomes (Doucey et al., 1998
), suggesting that C-mannosylation is involved in conventional glycosylation through the secretory pathway. This is consistent with the fact that almost all the C-mannosylated proteins found to date are secretory or membrane proteins. On the other hand, monomeric CMW was isolated from human urine (Gutsche et al., 1999
; Horiuchi et al., 1994
) and marine ascidians (Garcia et al., 2000
; Van Wagoner et al., 1999
), although the biological significance of the monomeric form is not known. It was reported that monomeric CMW in blood could be a novel biomarker of renal function (Takahira et al., 2001
). The functional relevance of glycosylation has been revealed in various cellular events, including cell development, growth, differentiation, and death (Dennis et al., 1999
; Haltiwanger and Lowe, 2004
; Lowe and Marth, 2003
; Varki, 1993
). However, the biological significance of C-mannosylation has yet to be fully investigated.
|
Here we describe the generation of specific antibodies against CMW. We also report the effect of hyperglycemic conditions on protein C-mannosylation in cultured cells and diabetic rat tissues. This is the first report concerning the characterization of protein C-mannosylation in the cell under pathological conditions such as diabetes mellitus.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Characterization of protein C-mannosylation in RAW264.7 cells cultured with low or high glucose concentrations
To investigate whether hyperglycemic conditions affect the level of protein C-mannosylation in cultured cells, mouse macrophage-like RAW264.7 cells were cultured with a low (5.5 mM) or high (30 mM) glucose medium for more than 5 days, and the C-mannosylation was examined immunologically. In Figure 4A, the expression level of CMW was examined by immunoblot analysis using the anti-CMW antibody in the cells cultured with low or high glucose concentrations. The level of C-mannosylation was increased in the hyperglycemic conditions especially in protein bands corresponding to 155, 76, 54, 52, and 35 kDa (arrows).
|
To investigate the mechanical link between the C-mannosylation and the activity of C-mannosyltransferase which is known to be responsible for the synthesis of C-mannosylated protein, the enzymatic activity was examined in the cell lysates from low- and high-glucose cultures. As shown in Figure 4B, the activity was increased under the hyperglycemic conditions rather than low-glucose conditions. These results suggest that the C-mannosyltransferase activity for CMW was increased in RAW264.7 cells cultured with a high concentration of glucose, resulting in the increased expression of C-mannosylated forms of specific cellular proteins under the hyperglycemic conditions. Next, the intracellular localization of C-mannosylated proteins was visualized in RAW264.7 cells by indirect immunofluorescence microscopy using the anti-CMW antibody (Figure 5). The immunoreactive signals showed a perinuclear vesicular pattern, and the intensity of the signals was enhanced in the cells cultured with high glucose rather than low glucose. To see the secretory vesicles, localization of clathrin heavy chain, a marker for secretory vesicles and the trans-Golgi network (Ihara et al., 2002), was also examined by indirect immunofluorescence microscopy. The immunoreactive signals of clathrin heavy chain showed a very similar localization to those of protein C-mannosylation, suggesting that C-mannosylated proteins might be localized in the secretory pathway of the cell.
|
Characterization of protein C-mannosylation in tissues of diabetic Zucker rats
To investigate whether hyperglycemia influences C-mannosylation in the tissues of those with diabetes, C-mannosylation was characterized immunologically in the male Zucker diabetic fatty (fa/fa) rat, an animal model of type II diabetes (Zucker and Antoniades, 1972). Nondiabetic Zucker lean (fa/+) rats were used as controls. Twenty-week-old rats were used for the study, because Zucker fatty rats exhibit hyperinsulinemia and hyperlipidemia at 6 weeks and become diabetic from 14 weeks (Coimbra et al., 2000
). Blood glucose levels for Zucker fatty, lean, and Sprague Dawley (another control) rats were 343.7 ± 22.5, 140.2 ± 35.0, and 152 ± 18.3 (mg/dl), respectively. Furthermore, blood insulin levels were 12.7 ± 1.5, 1.2 ± 0.4, and 0.9 ± 0.3 (ng/ml), respectively. These results indicated that Zucker fatty rats showed hyperglycemia and hyperinsulinemia at 20 weeks of age, whereas no symptoms were observed in lean and Sprague Dawley rats. This was also consistent with previous results (Coimbra et al., 2000
). As shown in Figure 6, the expression of C-mannosylated protein was examined in several tissues by immunoblot analysis using the anti-CMW antibody. In the tissue homogenates from brain, heart, liver, kidney, and serum, no significant difference was seen between Zucker fatty and lean rats in the level of C-mannosylation. In contrast, C-mannosylation was apparently increased in the aorta of Zucker fatty compared to lean rats. These results indicate that hyperglycemia increases the expression of C-mannosylated protein not in all tissues but in specific tissues, such as the aorta.
|
C-mannosylation is increased in the aortic vessel wall of Zucker fatty rats
To further characterize the expression of C-mannosylated protein in the aorta of Zucker fatty rats, C-mannosylation was examined immunohistochemically in the aortic vessel tissues using the anti-CMW antibody (Figure 7). The tissues were also counterstained with hematoxylin and eosin. In the aorta of Zucker fatty rats, no typical atherosclerotic change was observed compared to that of lean rats. However, the level of C-mannosylation was elevated in vessel walls of Zucker fatty compared to lean rats, and signals were diffusely seen in the intracellular and extracellular parts of whole layers of the vessel.
|
Recently, Stenina et al. (2003) reported that thrombospondin-1 (TSP-1) is induced in the aortic vessel wall of Zucker fatty rats, suggesting a pathogenic contribution of the increased expression of TSP-1 to the atherocsclerotic complications of blood vessels in diabetes. On the other hand, TSP-1 has been reported as a target of C-mannosylation (Hofsteenge et al., 2001
). Therefore, to investigate whether the expression of TSP-1 correlates with C-mannosylation, we also examined the expression of TSP-1 in the aortic vessel wall of Zucker fatty and lean rats. The expression of TSP-1 was elevated in the aortic vessel wall of Zucker fatty rats compared to lean rats (Figure 7), consistent with previous findings (Stenina et al., 2003
). The levels of TSP-1 expression and protein C-mannosylation in the aortic tissues were also compared by immunoblot analysis between Zucker fatty and lean rats using each specific antibody (Figure 8A). The results showed that for both antibodies, the intensity of immunoreactive bands was significantly elevated in the aortic tissue of Zucker fatty rats compared to that of lean rats. Notably, the specific bands of 190 and 170 kDa were likely to be identical between the immunoblots for CMW and TSP-1. To investigate whether TSP-1 is C-mannosylated in the aortic tissues of Zucker fatty rats, it was immunoprecipitated with the anti-TSP-1 antibody from the aortic tissue lysates of Zucker fatty rats, and then the immunoprecipitates were examined by immunoblotting using the anti-CMW antibody. As shown in Figure 8B, the immunoprecipitated TSP-1 corresponded to the bands of 190 and 170 kDa. These bands were also reactive with the anti-CMW antibody. Furthermore, the intensity of these bands was diminished in the presence of free CMW (1 mM). Together, these results indicate that TSP-1 was C-mannosylated in the aortic tissues of Zucker fatty rats.
|
Next, the C-mannosyltransferase activity was examined in tissue homogenates from the aorta of Zucker fatty and lean rats. For comparison, the aortic tissues from Sprague Dawley rats were also examined. As shown in Figure 8C, there was no significant difference in the C-mannosyltransferase activity observed in the tissue homogenates from Sprague Dawley and Zucker fatty and lean rats, despite the fact that the C-mannosylation was apparently increased in the aortic tissues of hyperglycemic Zucker fatty rats (Figure 7). Therefore, C-mannosylation occurred on TSP-1, and the increased expression of C-mannosylated protein was highly correlated with that of target proteins such as TSP-1 in the aortic vessel wall of Zucker fatty rats.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Core 2 GlcNAc transferase is a glycosyltransferase involved in the biosynthesis of O-glycan, and its up-regulation is implicated in the pathogenesis of diabetic complications (Nishio et al., 1995). The gene for core 2 GlcNAc transferase has been identified as being specifically up-regulated in rat diabetic cardiomyocytes (Nishio et al., 1995
). The up-regulation of core 2 GlcNAc transferase has been reported to enhance the actions of cytokines and induce a hypertrophic myocardium in transgenic mice, suggesting that specific changes to O-glycans have functional relevance in the pathogenesis of diabetic complications (Koya et al., 1999
). Furthermore, it has been reported that protein kinase Cdependent phosphorylation of core 2 GlcNAc transferase might be involved in the pathogenesis of diabetic retinopathy by mediating the increase in leukocyteendothelial cell adhesion and capillary occlusion (Chibber et al., 2003
). On the other hand, O-GlcNAc modification is a unique form of monoglycosylation of Ser or Thr residues in proteins and is also influenced by hyperglycemic conditions in vivo and in vitro (Hanover, 2001
; Parker et al., 2004
; Zachara and Hart, 2004
). An increase in the modification of O-GlcNAc was observed in pancreatic ß-cells of rats with streptozotocin-induced diabetes (Akimoto et al., 2000
; Hanover et al., 1999
; Liu et al., 2000
), rat aortic smooth muscle cells treated with high glucose (Akimoto et al., 2001
), and neonatal rat cardiomyocytes treated with high glucose (Clark et al., 2003
). In transgenic mice that overexpress O-GlcNAc transferase in adipose tissues and cardiac and skeletal muscles, insulin resistance and hyperleptinemia were induced (McClain et al., 2002
). In 3T3-L1 adipocytes, increased modification of O-GlcNAc by the inhibitor of N-acetylglucosaminidase resulted in insulin resistance associated with defects in protein kinase B/Akt activation (Vosseller et al., 2002
). Together, these results suggest a causative role for O-GlcNAc modifications in the development of insulin resistance and diabetic complications.
In contrast, it is not known whether hyperglycemia influences protein C-mannosylation. In the present study, we found that the level of C-mannosylation was elevated under hyperglycemic conditions in some cultured cells and diabetic tissues. In macrophage-like RAW264.7 cells, C-mannosylation was increased by hyperglycemic conditions, and the increase was concomitant with an increase in C-mannosyltransferase activity, suggesting that the promotion of C-mannosylation is due to the activation of C-mannosyltransferase under hyperglycemic conditions. We also examined whether C-mannosylation is induced by hyperglycemic conditions in other cell types such as Madin-Darby canine kidney cells. However, the up-regulation of protein C-mannosylation and C-mannosyltransferase activity was not observed in these cells cultured with a high glucose concentration (data not shown), suggesting that the up-regulation of C-mannosylation may not be common to all kinds of cells treated with high glucose. In RAW264.7 cells, the intracellular distribution of C-mannosylated proteins showed a perinuclear vesicular pattern, and was similar to that of clathrin heavy chain, a marker expressed in secretory vesicles and the trans-Golgi network. This strongly suggests that C-mannosylated proteins are part of the secretory pathway. The localization is also consistent with findings that C-mannosylated proteins are mainly secretory or membrane proteins, such as compliments, cytokines, and cytokine receptors (Furmanek and Hofsteenge, 2000).
The effect of hyperglycemic conditions on C-mannosylation was also examined in tissues of the Zucker fatty rat, a diabetic animal model. We found that protein C-mannosylation was specifically increased in the aortic tissues of Zucker fatty rats. However, there was no significant increase of C-mannosyltransferase activity in aortic tissue homogenates from Zucker fatty rats compared with controls. These results suggest that the increase of C-mannosylation in the diabetic aorta is not simply explained by the up-regulation of C-mannosyltransferase activity. Other possible explanations for the increase in C-mannosylation could be an increase in the synthesis of dolichol-P-mannose, a donor substrate for C-mannosylation (Doucey et al., 2000) or that of target proteins bearing the Trp-X-X-Trp motif. Sharma et al. (1987) reported that production of dolichol-P-mannose and dolichol-P-oligosaccharide was significantly down-regulated in liver slices from streptozotocin-treated diabetic rats. In contrast, an increase in the concentration of dolichol was observed in diabetic liver tissues of streptozotocin-treated rats, which suggests a dolichol-mediated enhancement of protein N-glycosylation by hyperglycemia (Bar-On et al., 1997
). Although it was not clear why conflicting results were obtained in the two studies concerning an altered dolichol metabolism in the liver of streptozotocin-treated rats, further investigation might be required to understand the correlation between C-mannosylation and the metabolism of dolichol in diabetic conditions.
To investigate the correlation between the levels of C-mannosylation and the expression of proteins targeted for C-mannosylation, we focused on the expression of TSP-1 in aortic tissues. TSPs are a family of multimeric, multidomain glycoproteins that function at the cell surface and in the extracellular matrix to regulate cellcell interactions and cellular signaling through binding with integral molecules such as TGF-ß, integrins, collagens, proteoglycans, CD36, CD47, calreticulin, and so forth (Adams, 2001; Elzie and Murphy-Ullrich, 2004
; Lawler, 2000
). TSP-1 is the best characterized of the TSPs, and its subunits are glycoylated with N-glycans (Furukawa et al., 1989
) and O-glycans (Hofsteenge et al., 2001
). TSP-1 is also C-mannosylated at the Trp-X-X-Trp motif in the TSP type 1 repeat (Hofsteenge et al., 2001
). In the present study, we found that TSP-1 was C-mannosylated in the aortic tissues of Zucker fatty rats, which was consistent with previous findings (Hofsteenge et al., 2001
). Furthermore, in immunohistochemical and immunoblot analyses, the levels for TSP-1 expression and protein C-mannosylation were concomitantly elevated in the aortic vessel of Zucker fatty rats compared to that of lean rats. Although the precise mechanism for the hyperglycemia-induced change of C-mannosylation is not yet clear, the different responses of C-mannosyltransferase activity seen in RAW264.7 cells and the aortic tissue of Zucker fatty rats suggest that the hyperglycemia-induced change of C-mannosylation is regulated differently in different types of cells or tissues. Collectively, the results obtained by using Zucker fatty rats suggest that the diabetes-induced increase of C-mannosylation in the aortic tissues is partly due to the diabetes-induced increase in the expression of target proteins such as TSP-1 to be C-mannosylated.
It has been reported that the expression of TSP-1 increased in aortic vessel walls of diabetic Zucker rats (Stenina et al., 2003). In that study, the authors suggested a relation between increased TSP-1 expression and accelerated atherosclerosis in the vascular tissues because TSP-1 is known to function in a variety of biological events, such as cell attachment, cell proliferation, angiogenesis, and apoptosis. Their findings are consistent with the results obtained in the present study. Atherosclerosis is a major vascular complication in diabetes (Semenkovich and Heinecke, 1997
), and the involvement of TSP-1 in the pathology of vascular impairment seems to be consistent with the high glucoseinduced up-regulation of TSP-1 expression in renal mesangial cells (Holmes et al., 1997
; McGregor et al., 1994
; Murphy et al., 1999
; Poczatek et al., 2000
; Tada and Isogai, 1998
; Wang et al., 2003
; Yevdokimova et al., 2001
) and renal tissues from Zucker fatty rats (Olson et al., 1999
).
In terms of the structural characteristics and functions of TSP-1, the Trp-X-X-Trp motif of the TSP type 1 repeat is believed to play an important role in the binding of TSP-1 with molecules such as glycosaminoglycans, TGF-ß, and matrix metalloproteases (Adams, 2001; Elzie and Murphy-Ullrich, 2004
). In fact, a study of the crystal structure of the TSP-1 type 1 repeats revealed that the Trps in the structure are oriented so that their polar atoms are exposed and available for potential ligand binding (Tan et al., 2002
). On the other hand, the Trp-X-X-Trp motif has also been revealed to be specifically modified by C-mannosylation in the case of TSP-1, suggesting some functional regulation of TSP-1 by C-mannosylation (Hofteenge et al., 2001; this study). Although how TSP-1 contributes to the diabetic complications is not well understood, the function of C-mannosylation may also need to be clarified to understand the functional regulation of TSP-1 in the pathogenesis.
Recently, chemically synthesized CMW and C-mannosylated peptides have become available for biomedical research (Manabe and Ito, 1999; Nishikawa et al., 1999
, 2001
). These chemicals and the specific antibody against CMW generated in this study are powerful tools for further investigation of the biological functions of C-mannosylation and C-mannosylated proteins in the cell.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synthesis of CMW
The CMW derivative, Fmoc-C2--D-C-mannosylpyranosyl-L-tryptophan, was synthesized as previously described (Manabe and Ito, 1999
). The purity and identity of the final product were verified by 1H nuclear magnetic resonance spectroscopy and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The protein chemical shifts and coupling constants are consistent with those reported before. The mass observed by MALDI mass spectrometry is consistent with the expected mass of the correct product.
Preparation with immunizing antigen
Fmoc-C2--D-C-mannosylpyranosyl-L-tryptophan was conjugated with KLH or ovalbumin by 1-ethyl-3-[3-dimethylamino-propyl]carbodiimide hydrochloride using the Imject Immunogen conjugation kit (Pierce Biotechnology, Rockford, IL), according to the manufacturers instructions. Fmoc was removed by alkaline treatment with 1 M sodium hydroxide and neutralized with hydrochloride. The sample was then desalted with gel filtration using a NAP-10 column (Amersham Pharmacia Biosciences, Little Chalfont, U.K.) and used as the immunizing antigen.
Generation and affinity purification of antibodies
Rabbits were immunized with KLH conjugated with CMW, then polyclonal antisera were generated according to the conventional protocol (Sigel et al., 1983). For affinity purification of the antibodies, CMW-conjugated affinity resins were prepared. The C-mannosylated peptide, C-Mannose-Trp-Ser-Pro-Trp-Cys, was synthesized as described before (Manabe and Ito, 1999
). The peptide was conjugated through sulfhydryl residues of Cys with SulfoLink coupling agarose using a SulfoLink kit (Pierce) according to the manufacturers directions. The antiserum was applied to the affinity resins and washed extensively with phosphate buffered saline (PBS, pH 7.2). The bound antibodies were eluted from the resins with 0.1 M glycine (pH 2.5), and neutralized by adding a 1/40 volume of 2 M Tris-hydrochloride (pH 8.5). The eluted solution was concentrated using a centricon (YM-10, Amicon) and then desalted with a gel filtration column equilibrated with PBS.
ELISA
One hundred microliters of a 2.5 µg/ml solution of ovalbumin conjugated with CMW in sodium carbonate buffer (pH 9.0) was added per well to a 96-well polystyrene microwell plate overnight at 4°C. The plate was washed with PBS and then blocked for 1 h with a 3% bovine serum albumin (BSA) in PBS. The wells were washed with PBS and incubated with 100 µl antibody solution for 2 h at room temperature. Then the wells were washed with PBS and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (Dako, Copenhagen) for 1 h at room temperature. After four washes with PBS, the wells were developed with a peroxidase substrate, TMB one solution (Promega, Madison, WI), and the absorbance at 450 nm was measured in a microplate reader.
Immunoblot analysis
Cultured cells were harvested and lysed in lysis buffer (20 mM Tris-hydrochloride [pH 7.2], 150 mM sodium chloride, and 1% Triton X-100 including protease inhibitors [20 µM APMSF, 50 µM pepstatin, 50 µM leupeptin]). After centrifugation at 8000 x g for 20 min, the supernatants were collected and used for the following assay. Protein samples were subjected to 5, 7.5, or 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) under reducing conditions and then transferred to a nitrocellulose membrane as described elsewhere (Ihara et al., 1997). The membrane was blocked with 5% skim milk in Tris-buffered saline (10 mM Tris-hydrochloride [pH 7.2] and 150 mM sodium chloride) and then incubated at 4°C overnight with the primary antibody in the blocking buffer. The blots were coupled with the peroxidase-conjugated secondary antibodies, washed, and then developed using the ECL chemiluminescence detection kit (Amersham Pharmacia Biosciences) according to the manufacturers instructions.
Cell culture
RAW264.7 cells were obtained from American Type Culture Collection. Cells were cultured in Dulbeccos modified Eagles medium containing 5.5 mM glucose, supplemented with 10% fetal calf serum under a humidified atmosphere of 95% air and 5% CO2 at 37°C. For hyperglycemic condition, the cells were cultured in Dulbeccos modified Eagles medium containing 30 mM glucose for more than 5 days.
Fluorescence microscopy
Cells (5 x 104/ml) were grown on Lab-Tek chamber slides (Nunc, Roskilde, Denmark) for 24 h. They were fixed with 4% paraformaldehyde in PBS and permeabilized for 10 min with PBS containing 1% Triton X-100. The cells were then blocked with 1% BSA in PBS, incubated with the antibody for 1 h, and washed with PBS containing 1% BSA. The immunoreactive primary antibodies were visualized with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit immunoglobulins (Cappel, Aurora, OH) or rhodamine-conjugated anti-mouse immunoglobulins (Dako). After a wash, the stained cells were mounted in the Vectashield medium. A Zeiss Axioskop2 (Carl Zeiss, Jena, Germanny), with epi-illumination for fluorescence, was used for the fluorescence microscopic analysis.
Assay of C-mannosyltransferase activity
The C-mannosyltransferase activity was assayed according to the methods of Doucey et al. (1998) with a slight modification. Enzyme assays with cell extracts were carried out in a 25 µl reaction volume containing 20 mM HEPES (pH 7.2), 110 mM potassium acetate, 2 mM magnesium acetate, 0.2% TritonX-100, 6.25 nmol of dolichol-P-[63H]mannose (40 Ci/mmol), and 0.9 mM of the substrate peptide for 2 h at 37°C. The reaction was stopped by adding 2 ml chloroform/methanol (3/2, vol/vol) and 0.48 ml water. After centrifugation, 0.2 ml of the upper phase containing the peptide was collected and then subjected to scintillation counting to determine the radioactivity.
Animals
Sprague Dawley, Zucker diabetic fatty (fa/fa), and lean (fa/+) rats were obtained from the Charles River Laboratory (Japan) and fed and housed in standard conditions at 22°C. The male rats at 20 weeks old were used for the experiments. Use of the animals was authorized according to the guidelines of the Declaration of Helsinki and the principles for the care and use of animals (Committee on Care and Use of Laboratory Animals of the Laboratory Animals Resources Commission on Life Sciences National Research Council [1985]. Guide for the Care and Use of Laboratory Animals, Public Health Service National Institutes of Health NIH Publication No. 86-23, Bethesda, MD).
Immunohistochemistry of rat aortic vessels
Tissues were embedded into paraffin, and sections were stained with the antibodies against cMW and TSP-1. Immunoreactive signals of primary antibodies were visualized with diaminobenzidine chromogenic substrate as described (Nakayama et al., 2004). For every section, a negative control with normal rabbit IgG was processed simultaneously. Sections were counterstained with hematoxylin and eosin.
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akimoto, Y., Kreppel, L.K., Hirano, H., and Hart, G.W. (2000) Increased O-GlcNAc transferase in pancreas of rats with streptozotocin-induced diabetes. Diabetologia, 43, 12391247.[CrossRef][ISI][Medline]
Akimoto, Y., Kreppel, L.K., Hirano, H., and Hart, G.W. (2001) Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation. Arch. Biochem. Biophys., 389, 166175.[CrossRef][ISI][Medline]
Bar-On, H., Nesher, G., Teitelbaum, A., and Ziv, E. (1997) Dolichol-mediated enhanced protein N-glycosylation in experimental diabetesa possible additional deleterious effect of hyperglycemia. J. Diabetes Complications, 11, 236242.[CrossRef][ISI][Medline]
Berry, E.M., Ziv, E., and Bar-On, H. (1980) Protein and glycoprotein synthesis and secretion by the diabetic liver. Diabetologia, 19, 535540.[ISI][Medline]
Chibber, R., Ben-Mahmud, B.M., Mann, G.E., Zhang, J.J., and Kohner, E.M. (2003) Protein kinase C ß2-dependent phosphorylation of core 2 GlcNAc-T promotes leukocyte-endothelial cell adhesion. A mechanism underlying capillary occlusion in diabetic retinopathy. Diabetes, 52, 15191527.
Clark, R.J., McDonough, P.M., Swanson, E., Trost, S.U., Suzuki, M., Fukuda, M., and Dillmann, W.H. (2003) Diabetes and the accompanying hyperglycemia impairs cardiomyocyte calcium cycling through increased nuclear O-GlcNAcylation. J. Biol. Chem., 278, 4423044237.
Coimbra, T.M., Janssen, U., Grone, H.J., Ostendorf, T., Kunter, U., Schmidt, H., Brabant, G., and Floege, J. (2000) Early events leading to renal injury in obese Zucker (fatty) rats with type II diabetes. Kidney Int., 57, 167182.[CrossRef][ISI][Medline]
De Peredo, A.G., Klein, D., Macek, B., Hess, D., Peter-Katalinic, J., and Hofsteenge, J. (2002) C-mannosylation and O-fucosylation of thrombospondin type 1 repeats. Mol. Cell. Proteomics, 1, 1118.
Dennis, J.W., Granovsky, M., and Warren, C.E. (1999) Protein glycosylation in development and disease. BioEssays, 21, 412421.[CrossRef][ISI][Medline]
Doucey, M.-A., Hess, D., Cacan, R., and Hofsteenge, J. (1998) Protein c-mannosylation is enzyme-catalysed and uses dolichyl-phosphate-mannose as a precursor. Mol. Biol. Cell, 9, 291300.
Elzie, C.A. and Murphy-Ullrich, J.E. (2004) The N-terminus of thrombospondin: the domain stands apart. Int. J. Biochem. Cell Biol., 36, 10901101.[CrossRef][ISI][Medline]
Furmanek, A. and Hofsteenge, J. (2000) Protein c-mannosylation: facts and questions. Acta Biochim. Polonica., 47, 781789.[ISI][Medline]
Furmanek, A., Hess, D., Rogniaux, H., and Hofsteenge, J. (2003) The WSAWS motif is c-hexosylated in a soluble form of the erythropoietin receptor. Biochemistry, 42, 84528458.[CrossRef][ISI][Medline]
Furukawa, K., Roberts, D.D., Endo, T., and Kobata, A. (1989) Structural study of the sugar chains of human platelet thrombospondin. Arch. Biochem. Biophys., 270, 302312.[CrossRef][ISI][Medline]
Garcia, A., Lenis, L.A., Jimenez, C., Debitus, C., Quinoa, E., and Riguera, R. (2000) The occurrence of the human glycoconjugate C(2)--D-mannosylpyranosyl-L-tryptophan in marine ascidians. Org. Lett., 2, 27652767.[CrossRef][ISI][Medline]
Gutsche, B., Grun, C., Scheutzow, D., and Herderich, M. (1999) Tryptophan glycoconjugates in food and human urine. Biochem. J., 343, 1119.[CrossRef][ISI][Medline]
Haltiwanger, R.S. and Lowe, J.B. (2004) Role of glycosylation in development. Annu. Rev. Biochem., 73, 491537.[CrossRef][ISI][Medline]
Hanover, J.A. (2001) Glycan-dependent signaling: O-linked N-acetylglucosamine. FASEB J., 15, 18651876.
Hanover, J.A., Lai, Z., Lee, G., Lubas, W.A., and Sato, S.M. (1999) Elevated O-linked N-acetylglucosamine metabolism in pancreatic ß-cells. Arch. Biochem. Biophys., 362, 3845.[CrossRef][ISI][Medline]
Hartmann, S. and Hofsteenge, J. (2000) Properdin, the positive regulator of complement, is highly c-mannosylated. J. Biol. Chem., 275, 2856928574.
Hofsteenge, J., Muller, D.R., de Beer, T., Loffler, A., Richter, W.J., and Vliegenthart, J.F. (1994) New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us. Biochemistry, 33, 1352413530.[ISI][Medline]
Hofsteenge, J., Huwiler, K.G., Macek, B., Hess, D., Lawler, J., Mosher, D.F., and Peter-Katalinic, J. (2001) C-mannosylation and O-fucosylation of the thrombospondin type 1 module. J. Biol. Chem., 276, 64856498.
Holmes, D.I.R., Wahab, N.A., and Mason, R.M. (1997) Identification of glucose-regulated genes in human mesangial cells by mRNA differentiation display. Biochem. Biophy. Res. Commun., 238, 179184.[CrossRef][ISI][Medline]
Horiuchi, K., Yonekawa, O., Iwahara, K., Kanno, T., Kurihara, T., and Fujise, Y. (1994) A hydrophilic tetrahydro-ß-carboline in human urine. J. Biochem. (Tokyo), 115, 362366.[Abstract]
Ihara, Y., Sakamoto, Y., Mihara, M., Shimizu, K., and Taniguchi, N. (1997) Overexpression of N-acetylglucosaminyltransferase III disrupts the tyrosine phosphorylation of Trk with resultant signaling dysfunction in PC12 cells treated with nerve growth factor. J. Biol. Chem., 272, 96299634.
Ihara, Y., Yasuoka, C., Kageyama, K., Wada, Y., and Kondo, T. (2002) Tyrosine phosphorylation of clathrin heavy chain under oxidative stress. Biochem. Biophys. Res. Commun., 297, 353360.[CrossRef][ISI][Medline]
Koya, D., Dennis, J.W., Warren, C.E., Takahara, N., Schoen, F.J., Nishio, Y., Nakajima, T., Lipes, M.A., and King, G.L. (1999) Overexpression of core 2 N-acetylglucosaminyltransferase enhances cytokine actions and induces hypertrophic myocardium in transgenic mice. FASEB J., 13, 23292337.
Kornfeld, R. and Kornfeld, S. (1985) Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 54, 631664.[CrossRef][ISI][Medline]
Lawler, J. (2000) The functions of thrombospondin-1 and 2. Curr. Opin. Cell Biol., 12, 634640.[CrossRef][ISI][Medline]
Liu, K., Paterson, A.J., Chin, E., and Kudlow, E. (2000) Glucose stimulates protein modification by O-linked GlcNAc in pancreatic ß cells: linkage of O-linked GlcNAc to ß cell death. Proc. Natl Acad. Sci. USA, 97, 28202825.
Lowe, J.B. and Marth, J.D. (2003) A genetic approach to mammalian glycan function. Annu. Rev. Biochem., 72, 643691.[CrossRef][ISI]
Manabe, S. and Ito, Y. (1999) Total synthesis of novel subclass of glyco-amino acid structure motif: C2--C-mannosylpyranosyl-L-tryptophan. J. Am. Chem. Soc., 121, 97549755.[CrossRef][ISI]
McClain, D.A., Lubas, W.A., Cooksey, R.C., Hazel, M., Parker, G.J., Love, D.C., and Hanover, J.A. (2002) Altered glycan-dependent signaling induces insulin resistance and hyperleptinemia. Proc. Natl Acad. Sci. USA, 99, 1069510699.
McGregor, B., Colon, S., Mutin, M., Chignier, E., Zech, P., and McGregor, J. (1994) Thrombospondin in human glomerulopathies. A marker of inflammation and early fibrosis. Am. J. Pathol., 144, 12811287.[Abstract]
Murphy, M., Godson, C., Cannon, S., Kato, S., Mackenzie, H.S., Martin, F., and Brady, H.R. (1999) Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. J. Biol. Chem., 274, 58305834.
Nakayama, T., Yoshizaki, A., Kawahara, N., Ohtsuru, A., Wen, C.Y., Fukuda, E., Nakashima, M., and Sekine I. (2004) Expression of Tie-1 and 2 receptors, and angiopoietin-1, 2 and 4 in gastric carcinoma; immunohistochemical analyses and correlation with clinicopathological factors. Histopathology, 44, 232239.[CrossRef][ISI][Medline]
Nishikawa, T., Ishikawa, M., and Isobe, M. (1999) Synthesis of -1-C-mannosyltryptophan derivative, naturally occurring C-glycosyl amino acid found in human ribonuclease. Synlett., 123125.
Nishikawa, T., Ishikawa, M., Wada, K., and Isobe, M. (2001) Total synthesis of a 1-C-mannosyltryptophan, a naturally occurring C-glycosyl amino acid. Synlett., 945947.
Nishio, Y., Warren, C.E., Buczek-Thomas, J.A., Rulfs, J., Koya, D., Aiello, L.P., Feener, E.P., Miller, T.B., Dennis, J.W., and King, G.L. (1995) Identification and characterization of a gene regulating enzymatic glycosylation which is induced by diabetes and hyperglycemia specifically in rat cardiac tissue. J. Clin. Invest., 96, 17591767.[ISI][Medline]
Olson, B.A., Day, J.R., and Lapling, N.J. (1999) Age-related expression of renal thrombospondin 1 mRNA in F344 rats: resemblance to diabetes-induced expression in obese Zucker rats. Pharmacology, 58, 200208.[CrossRef][ISI][Medline]
Parker, G., Taylor, R., Jones, D., and McClain, D. (2004) Hyperglycemia and inhibition of glycogen synthase in streptozotocin-treated mice. Role of O-linked N-acetylglucosamine. J. Biol. Chem., 279, 2063620642.
Perez-Vilar, J., Randell, S.H., and Boucher, R.C. (2004) C-mannosylation of MUC5AC and MUC5B Cys subdomains. Glycobiology, 14, 325337.
Poczatek, M.H., Hugo, C., Darley-Usmar, V., and Murphy-Ullrich, J.E. (2000) Glucose stimulation of transforming growth factor-ß bioactivity in mesangial cells is mediated by thrombospondin-1. Am. J. Pathol., 157, 13531363.
Schachter, H. and Brockhausen, I. (1989) The biosynthesis of branched O-glycans. Symp. Soc. Exp. Biol., 43, 126.[Medline]
Semenkovich, C.F. and Heinecke, J.W. (1997) The mystery of diabetes and athrosclerosis. Time for a new prot. Diabetes, 46, 327334.[Abstract]
Sharma, C., Dalferes, E.R. Jr., Radhakrishnamurthy, B., DePaolo, C.J., and Berenson, G.S. (1987) Hepatic glycoprotein synthesis in streptozotocin diabetic rats. Biochem. Int., 15, 395401.[ISI][Medline]
Sigel, M.B., Sinha, Y.N., and VanderLaan, W.P. (1983) Production of antibodies by inoculation into lymph nodes. Methods Enzymol., 93, 312.[ISI][Medline]
Spiro, R.G. (2002) Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology, 12, 43R56R.
Spiro, R.G. and Spiro, M.J. (1971) Effect of diabetes on the biosynthesis of the renal glomerular basement membrane. Studies on the glucosyltransferase. Diabetes, 20, 641648.[ISI][Medline]
Stenina, O.I., Krukovets, I., Wang, K., Zhou, Z., Forudi, F., Penn, M.S., Topol, E.J., and Plow, E.F. (2003) Increased expression of thrombospondin-1 in vessel wall of diabetic zucker rat. Circulation, 107, 32093215.
Tada, H. and Isogai, S. (1998) The fibronectin production is increased by thrombospondin via activation of TGF-ß in cultured human mesangial cells. Nephron, 79, 3843.[CrossRef][ISI][Medline]
Takahira, R., Yonemura, K., Yonekawa, O., Iwahara, K., Kanno, T., Fujise, Y., and Hashida, A. (2001) Tryptophan glycoconjugate as a novel marker of renal function. Am. J. Med., 110, 192197.[CrossRef][ISI][Medline]
Tan, K., Duquette, M., Liu, J.-H., Dong, Y., Zhang, R., Joachimiak, A., Lawler, J., and Wang, J.-H. (2002) Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication. J. Cell Biol., 159, 373382.
Tepperman, H.M., DeWitt, J., and Tepperman, J. (1983) The effects of streptozotocin diabetes on the activities of rat liver glycosyltransferases. Diabetes, 32, 412415.[Abstract]
Van Wagoner, R.M., Jompa, J., Tahir, A., and Ireland, C.M. (1999) Trypargine alkaloids from a previously undescribed eudistoma sp. ascidian. J. Nat. Prod., 62, 794797.[CrossRef][ISI][Medline]
Varki, A. (1993) Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 3, 97130.[Abstract]
Vosseller, K., Wells, L., Lane, M.D., and Hart, G.W. (2002) Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes. Proc. Natl Acad. Sci. USA, 99, 53135318.
Wang, S., Wu, X., Lincoln, T.M., and Murphy-Ullrich, J.E. (2003) Expression of constitutively active cGMP-dependent protein kinase prevents glucose stimulation of thrombospondin 1 expression and TGF-b activity. Diabetes, 52, 21442150.
Yevdokimova, N., Wahab, A.N., and Mason, R.M. (2001) Thrombospondin-1 is the key activator of TGF-ß1 in human mesangial cells exposed to high glucose. J. Am. Soc. Nephrol., 12, 703712.
Zachara, N.E. and Hart, G.W. (2004) O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress. Biochim. Biophys. Acta, 1673, 1328.[ISI][Medline]
Zucker, L.M. and Antoniades, H.N. (1972) Insulin and obesity in the Zucker genetically obese rat ìfattyî. Endocrinology, 90, 13201330.[ISI][Medline]