Structural characterization of N-linked oligosaccharides of laminin from rat kidney: changes during diabetes and modulation by dietary fiber and butyric acid Adishesha Puneeth Kumar, Chilkunda D. Nandini and Paramahans V. Salimath Department of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore, India Introduction Changes in oligosaccharide structures are associated with many physiological and pathological events. The biochemical role of oligosaccharides in glycoproteins has been examined for intracellular trafficking, protein- ase susceptibility, molecular conformation, protein sta- bility and protein–protein interactions [1]. Laminin has been shown to be a glycoprotein with N-linked oligo- saccharides and these oligosaccharides exhibit various biological functions [2,3]. To date, 74 N-glycosylation sites in laminin isolated from mouse have been reported [2]. Fifteen isoforms of laminin have been identified, wherein kidney laminin belongs to isoform 11 with alpha-5-, beta-3- and gamma-3-type chains [4]. Laminin in kidney plays an important role in main- taining the structural integrity of the glomerular base- ment membrane [5]. During the past decade, the inci- dence of end-stage renal disease as a result of diabetes has risen dramatically due to sustained hyperglycemia. In patients with diabetes, persistent hyperglycemic status is reported to cause nonenzymatic addition of carbohydrate moieties to laminin in kidney, resulting in the formation of advanced glycated end products. These abnormally glycated proteins create remarkable shape changes and deformations, which reduce the ability of laminin to polymerize, leading to compro- mised interactions between laminin and other basement Keywords diabetes; kidney; laminin; MALDI-TOF; oligosaccharides Correspondence P. V. Salimath, Dept. of Biochemistry and Nutrition, Central Food Technological Research Institute, Mysore – 570 020, Karnataka, India Fax: +91 821 251 7233 Tel: +91 821 251 4876 E-mail: salimath1954@gmail.com (Received 14 June 2010, revised 28 September 2010, accepted 26 October 2010) doi:10.1111/j.1742-4658.2010.07940.x Carbohydrates of laminin, a family of large multidomain glycoproteins, have been implicated in various cellular activities including maintaining the protein structure, its function and also basement membrane integrity. Dur- ing the course of our investigation, we observed that purified laminin from kidneys of control, diabetic, and dietary fiber- and butyric acid-treated dia- betic rats showed differences in binding to extracellular matrix components. This prompted us to determine whether there are structural changes in lam- inin oligosaccharides. In this study, we have characterized a few major N-linked oligosaccharides isolated from purified laminin in various experi- mental groups, viz. normal, diabetic and diabetic rats fed with dietary fiber and butyric acid. Sugar composition, as identified by GLC, revealed the presence of mannose, galactose and N-acetylglucosamine. In order to study fine structures of the oligosaccharides, N-linked oligosaccharides of laminin were released by Peptide-N-glycosidase F digestion, end-labeled with 2-anthranilic acid and fractionated by lectin affinity chromatography. Furthermore, structural elucidation carried out by MALDI-TOF MS ⁄ MS analysis showed variations in the oligosaccharide sequence of laminin during diabetes which were altered by the feeding of dietary fiber and butyric acid. Abbreviations 2AA, 2-anthranilic acid; FFC, dietary fiber-fed control; FFD, dietary fiber-fed diabetic; FFD-500, dietary fiber-fed diabetic with 500 mg butyric acid; PGNase-F, Peptide-N-glycosidase F; SFC, starch-fed control; SFD, starch-fed diabetic. FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS 143 membrane macromolecules [6]. Nonenzymatic glyca- tion also affects the balance between the synthesis and degradation of laminin in kidney. We have previ- ously observed that the laminin content decreases dur- ing diabetes and the binding property of purified laminin to various extracellular matrix components becomes altered (A. P. Kumar, C. D. Nandini & P. V. Salimath, unpublished data). Hence, for proper func- tioning of the kidney, maintaining the glomerular base- ment membrane architecture is crucial, which in turn depends on the proper structure and function of matrix proteins, including laminin and its oligosaccha- ride moieties. Knowledge about the nature of oligosaccharides pres- ent in the glycoproteins is crucial for better understand- ing of its functional aspects [7]. However, structural characterization of oligosaccharides in glycoproteins is one of the most difficult challenges and often requires the application of multiple analytical approaches [8]. MS has become a powerful analytical tool for the struc- tural characterization of oligosaccharides. Of several MS techniques, the soft ionization method MALDI- TOF MS has been used extensively in recent years to determine oligosaccharide structures [9] and this approach has proved to be advantageous and also very sensitive in elucidating the structural sequences of oligo- saccharides [10]. Derivatization of oligosaccharides at their reducing end with chromophores or fluorophores to increase absorption, fluorescence or MS sensitivity has become a common practice in recent years [11]. Hence, in this study an attempt was made to carry out the structural elucidation of high mannose containing N-linked oligosaccharides of laminin from different experimental groups by facile labeling of oligosaccha- rides with 2-anthranilic acid (2AA) [7] in conjunction with MALDI-TOF MS ⁄ MS analysis to obtain their fine structural details. Dietary intervention is a potential strategy in the prevention and treatment of many metabolic disor- ders, including diabetes [12]. Butyric acid (CH 3 CH 2 CH 2 COOH), a four-carbon short-chain fatty acid is produced in large amounts from dietary fibers after their fermentation in the large intestine, along with other short-chain fatty acids like acetate and propio- nate [13]. The bioactivities of butyric acid are related to its ability to modify nuclear architecture and induce apoptosis, changing the structure of chromatin through its effect on post-translation modification, modulation of the activities of key regulatory enzymes involved in metabolic activities, and also its ability to regulate the gene expression [13]. Our earlier studies have shown that supplementing the diet with dietary fiber and butyric acid was beneficial in ameliorating the diabetic condition of rats [14]. In this article, we have tried to determine and explore the changes in N- linked oligosaccharides of laminin during diabetes and the likelihood of dietary fiber and butyric acid in mod- ulating these changes. Results Rats experimentally induced with diabetes using strep- tozotocin were used for the study. Age-matched rats which were injected with buffer served as controls. Animals were fed with different experimental diets for 65 days [15]. All diabetic rats were hyperglycemic at the end of experimental period and, as expected, showed increased urine output, excretion of sugar and increase in glomerular filtration rate. Feeding dietary fiber and butyric acid resulted in the amelioration of the above parameters to a considerable extent (Table 1). Identification of monosaccharides by GLC As a first step, laminin from rat kidneys from different experimental groups was purified and purity was ascer- tained by SDS ⁄ PAGE using silver nitrate staining (depicted in Fig. S1 for laminin from starch-fed con- trols as a representative). This showed bands corre- sponding to 400 and 200 kDa which are characteristic of laminin. Composition analysis of laminin was car- ried out in terms of total sugars, amino sugars and sia- lic acid (Table 2). The monosaccharide composition of oligosaccharides of laminin was determined by GLC after acid hydrolysis and was carried out for laminin purified from starch-fed control (SFC) and starch-fed diabetic (SFD) groups as a representative. The results of composition analysis and GLC revealed the presence of neutral sugars like mannose and galactose and amino sugar as N-acetylglucos- amine, along with sialic acid which accounted for  22 and 35% of total carbohydrate content in SFC and SFD groups, respectively. Purification of 2AA-labeled oligosaccharides from different experimental groups The low yield of laminin from rat kidney cortex necessi- tated a sensitive method for the purification and subse- quent characterization of laminin oligosaccharides. Hence, in this study laminin oligosaccharides, which were released by Peptide-N-glycosidase F (PNGase-F) digestion, were tagged with 2AA to increase the sensitiv- ity of detection. The 2AA-labeled oligosaccharides were then fractionated using Concanavalin-A–Sepharose Laminin oligosaccharide changes during diabetes A. P. Kumar et al. 144 FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS lectin-affinity chromatography. The oligosaccharides from all the experimental groups that bound to Conca- navalin-A–Sepharose lectin were eluted as two major peaks with 0.2 and 0.3 m a-methyl d-glucopyranoside, which indicated the presence of at least two types of high mannose containing oligosaccharides in laminin (Fig. 1). Fractions eluting with 0.4 and 0.5 m a-methyl d-glucopyranoside were in minor amounts and so were not taken up for further analysis. MALDI-TOF MS/MS analysis of purified oligosaccharide Affinity-eluted 2AA-derivatized oligosaccharides of laminin from different experimental groups were fur- ther analyzed by subjecting them to MALDI-TOF MS. Each of the fractions showed signals characteristic of the oligosaccharides. In the control group, the 0.2 m eluted fraction showed a signal with an m ⁄ z value of 2185 (Fig. 2A) and the 0.3 m eluted fraction had signal at 3240 (m ⁄ z)(Fig. 3A). In the diabetic group, the 0.2 Table 1. Effect of dietary fiber and butyric acid on fasting blood sugar, urine sugar, urine volume and glomerular filtration rate (GFR) in diabetic rats. Values are the mean ± SEM of six controls and 10 diabetic rats. SFC, starch-fed control; FFC, dietary fiber-fed control; SFD, starch-fed diabetic; FFD, dietary fiber-fed diabetic; FFD-500, dietary fiber and butyric acid-fed diabetic. Group FBS (mgÆdL )1 ) Urine sugar (gÆday )1 ) Urine output (mLÆday )1 ) GFR (mLÆmin )1 ) SFC 108.45 ± 3.1 0.19 ± 0.01 16.3 ± 3.3 0.83 ± 0.04 SFD 348.61 ± 3.6 a 7.35 ± 0.50 a 73.5 ± 5.3 a 3.98 ± 0.45 a FFC 106.54 ± 5.2 0.18 ± 0.03 18.3 ± 3.3 1.08 ± 0.05 FFD 264.87± 2.5 b 5.56 ± 0.30 b 55.6 ± 3.4 b 2.48 ± 1.01 b FFD-500 188.75 ± 2.3 b 4.72 ± 0.20 b 47.2 ± 2.9 b 2.01 ± 0.03 b a Statistically significant at P < 0.05 when compared with SFC. b Statistically significant at P < 0.05 when compared with SFD. Table 2. Effect of dietary fiber and butyric acid on total sugar, amino sugar and sialic acid content of laminin (lgÆ100 lg )1 protein). Values are average of duplicate analyses carried out on laminin puri- fied from pooled control (6) and diabetic (10) rats. SFC, starch-fed control; FFC, dietary fiber-fed control; SFD, starch-fed diabetic; FFD, dietary fiber-fed diabetic; FFD-500, dietary fiber and butyric acid-fed diabetic. Group Total sugar Amino sugar Sialic acid SFC 21.91 ± 1.16 2.91 ± 0.31 2.15 ± 0.15 SFD 34.81 ± 1.57 5.11 ± 1.20 3.43 ± 0.23 FFC 21.57 ± 0.67 2.75 ± 0.34 2.18 ± 0.11 FFD 28.14 ± 1.42 4.08 ± 0.53 2.95 ± 0.35 FFD-500 25.74 ± 0.43 3.43 ± 0.44 2.58 ± 0.23 0 111213141 0.2 M 0.2 M 0.3 M 0.2 M 0.3 M 0.2 M 0.3 M 0.3 M 51 Fraction no. 61 1 1121314151 Fraction no. 61 1 1 6 11 16 2621 31 36 4641 5651 61 11 21 31 41 51 Fraction no. Fraction no. 61 0.05 0.1 A at 333 nm A at 333 nm 0.15 0.2 AB CD 0 0.05 0.1 0.15 0.2 0.25 A at 333 nm A at 333 nm 0 0.05 0.1 0.15 0.2 0.25 0.5 0.4 0.3 0.2 0.1 0 Fig. 1. Purification of 2AA-labeled oligosac- charides. Oligosaccharides obtained by PNGase-F digestion were end-labeled with 2AA and purified on a lectin–agarose column using D-glucopyranoside as the eluent. Elution profiles are shown by horizontal bars with numbers representing the concentra- tion of glucopyranoside. (A) Control group (SFC ⁄ FFC), (B) diabetic group (SFD), (C) dietary fiber-fed diabetic group (FFD), (D) dietary fiber and butyric acid-fed group (FFD-500). A. P. Kumar et al. Laminin oligosaccharide changes during diabetes FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS 145 and 0.3 m eluted fractions showed signals with masses of 3322 (m ⁄ z ) and 3443 (m ⁄ z), respectively (Figs4A and 5A). Similarly, in the fiber-treated diabetic group the 0.2 and 0.3 m eluted fractions showed signals with masses of 2713 (m ⁄ z) and 3119 (m ⁄ z), respectively (Figs 6A and 7A). Furthermore, the MALDI-TOF spectrum of 0.2 and 0.3 m eluted fractions in the fiber plus butyric acid-treated diabetic group showed signals with masses of 2389 (m ⁄ z)(Fig. 8A) and 2592 (m ⁄ z) (Fig. 9A), respectively. An attempt was made to deduce their fine structures by further subjecting them to MS ⁄ MS. Peak 1 (0.2 m eluted) of the control (SFC) group, when subjected to MS ⁄ MS with an m ⁄ z value of 2185 split into five fragments with an m ⁄ z values ranging from 566 to 2185 (Fig. 2B). These are due to oligomers of DP- 2[m⁄ z 566 (M + Na + + 2AA)], DP-5 [m ⁄ z 1052 (M + Na + + 2AA)], DP-7 [m ⁄ z 1376 (M + Na + + 2AA)], DP-11 [m ⁄ z 2024 (M + Na + + 2AA)] and DP-12 [m ⁄ z 2185 (M + Na + + 2AA)]. peak 2 (0.3 m-eluted) of the control (SFC) group with an m ⁄ z value of 3240 was split into six fragments (Fig. 3B) which are identified as oligo- mers of DP-2 [m ⁄ z 566 (M + Na + + 2AA)], DP-4 [m ⁄ z 890 (M + Na + + 2AA)], DP-8 [m ⁄ z 1620 (M + Na + + 2AA)], DP-12 [m ⁄ z 2268 (M + Na + + 2AA)], DP-16 [m ⁄ z 2916 (M + Na + + 2AA)] and DP-18 [m ⁄ z 3240 (M + Na + + 2AA)]. Similarly, peak 1 of the diabetic (SFD) group with an m ⁄ z value of 3322 was split into eight major fragments (Fig. 4B) which are identified as oligomers of DP-2 [m ⁄ z 566 (M + Na + + 2AA)], DP-4 [m ⁄ z 889 (M + Na + + 2AA)], DP-6 [m ⁄ z 1296 (M + Na + + 2AA)], DP-8 [m ⁄ z 1702 (M + Na + + 2AA)], DP-11 [m ⁄ z 2188 (M + Na + + 2AA)], DP-13 [m ⁄ z 2512 (M +Na + + 2AA)], DP-15 [m ⁄ z 2836 (M + Na + + 2AA)], and DP-18 [m ⁄ z 3323 (M + Na + + 2AA)]. peak 2 of the diabetic (SFD) group with an m ⁄ z value of 3443 was split into seven major fragments (Fig. 5B) which are identified as oligomers of DP-2 [m ⁄ z 566 (M + Na + + 2AA)], DP-5 [m ⁄ z 1092 (M + Na + + 2AA)], DP-8 [m ⁄ z 1620 (M + Na + + 2AA)], DP-11 [m ⁄ z 2148 (M + Na + + 2AA)], DP-14 [m ⁄ z 2633 (M + Na + + 2AA)], DP-16 [m ⁄ z 2956(M + Na + + 2AA)] and DP-19 [m ⁄ z 3443 (M + Na + + 2AA)]. Similarly, peak 1 of the fiber-fed diabetic (FFD) group with an m ⁄ z value of 2713 was split into six major fragments (Fig. 6B) which are identified as oligomers of DP-2 [m ⁄ z 566 (M + Na + + 2AA)], DP-5 [m ⁄ z 1052 (M + Na + + 2AA)], DP-8 [m ⁄ z 1579 (M + Na + + 2AA)], DP-11 [m ⁄ z 2064 (M + Na + +2AA)], DP-13 0 0 100 200 1000 500 700 900 1100 1300 1500 1700 1900 2100 2300 1500 2000 2500 3000 3500 2185.34 A B 566.56 1052.43 1376.93 2024.18 2185.84 4000 4500 5000 5500 m/z m/z 1000 2000 3000 Intens. [a.u.] Intens. [a.u.] Fig. 2. Oligosaccharide moieties of laminin released by PNGase-F. Oligosaccharide moi- eties were labeled with 2AA and seperated by lectin–agarose chromatography using stepwise elution. Eluted oligosaccharides were then subjected to MALDI-TOF MS ⁄ MS analysis for structural elucidation as detailed in the Materials and Methods. MALDI-TOF spectra of peak 1 (0.2 M-eluted) from the SFC group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). Laminin oligosaccharide changes during diabetes A. P. Kumar et al. 146 FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS [m ⁄ z 2389 (M + Na + + 2AA)] and DP-15 [m ⁄ z 2713 (M + Na + + 2AA)]. peak 2 of the fiber-fed diabetic (FFD) group with an m ⁄ z value of 3119 was split into five major fragments (Fig. 7B) which are identified as oligomers of DP-2 [m ⁄ z 566 (M + Na + + 2AA)], DP-6 [m ⁄ z 1213 (M + Na + + 2AA)], DP-10 [m ⁄ z 1903 (M + Na + + 2AA)], DP-13 [m ⁄ z 2471 (M + Na + +2AA)] and DP-17 [m ⁄ z 3119 (M + Na + + 2AA)]. Further, peak 1 of the fiber plus butyric acid-fed diabetic (FFD-500) group with an m ⁄ z value of 2389 was fragmented into five major fragments (Fig. 8B) which are identified as oligomers of DP-3 [m ⁄ z 728 (M + Na + + 2AA)], DP-5 [m ⁄ z 1093 (M + Na + + 2AA)], DP-7 [m ⁄ z 1417 (M + Na + + 2AA)], DP- 10 [m ⁄ z 1903 (M + Na + + 2AA)] and DP-13 [m ⁄ z 2389 (M + Na + + 2AA)]. peak 2 of the fiber plus butyric acid fed diabetic (FFD-500) group with an m ⁄ z value of 2592 was fragmented into four major peaks (Fig. 9B) which are identified as oligomers of DP-4 [m ⁄ z 890 (M + Na + + 2AA)], DP-8 [m ⁄ z 1579 (M + Na + + 2AA)], DP-11 [m ⁄ z 2055 (M + Na + + 2AA)] and DP-14 [m ⁄ z 2592 (M + Na + + 2AA)]. Based on the spectral signals, oligosaccharide struc- tures were constructed, the proposed structures of which are given in Table 3. Discussion Isolation of single molecular species of oligosaccha- rides from laminin has remained a challenge because of their tremendous heterogeneity [16]. In addition, they are present in tissues such as kidney in minor amounts, as a result of which large pools of tissues are required to isolate and characterize them. Although reports are available on laminin oligosaccha- rides in Engelbreth–Holm–Swarm tumor cells [17], information is lacking with respect to kidney laminin. In pathological conditions such as diabetes, quantita- tive changes in laminin content have been observed [6] and our own observations have shown that there is a difference in binding of laminin purified from control, diabetic and fiber- and butyric acid-treated groups towards extracellular matrix components such as type IV collagen, fibronectin and heparan sulfate (A. P. Kumar, C. D. Nandini & P. V. Salimath, unpub- lished data). In this article, therefore, an attempt was made to isolate and characterize N-linked oligosaccha- rides which bound to Concanavalin-A–Sepharose from control and diabetic rat kidney and determine whether dietary fiber and butyric acid treatment results in changes in these oligosaccharides. Interest in the 0 0 500 566.91 890.11 1620.47 2268.38 2916.24 3240.14 3240.24 1000 1500 2000 2500 3000 3500 Mass 100 % 1000 1500 25002000 35003000 45004000 5500 m/z 5000 1000 2000 3000 A B Intens. [a.u.] Fig. 3. MALDI-TOF spectra of peak 2 (0.3 M-eluted) from the SFC group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Figs 1 and 2. A. P. Kumar et al. Laminin oligosaccharide changes during diabetes FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS 147 complexities of oligosaccharide structure on glycopro- teins is increasing because of their pivotal functions in cell adhesion and recognition mechanisms, and their importance in signal transduction and as markers of differentiation and carcinogenesis [18]. Hence, studying the structure of oligosaccharides is crucial in ascertain- ing their role. The total carbohydrate content in laminin was shown to be  32%. Reports of the abundance of car- bohydrates in laminin differ between the research groups. Reports of high (30%) [16] and low (12%) [2] levels of carbohydrates are available. Our report is in agreement to values reported by Knibbs et al. [16]. Our study showed the presence of only N-acetylglucos- amine, which is in agreement with the earlier reports demonstrating that laminin has predominantly N-linked oligosaccharides [16]. Higher amounts of total carbohydrates were observed in laminin from dia- betic groups. This may be because of nonenzymatic glycation as a result of sustained hyperglycemia [19]. During prolonged hyperglycemia, extracellular matrix proteins undergo glycosylation through Amadori rear- rangement, followed by further rearrangement and epi- merization reactions, which probably involve various enol intermediates that might lead to the formation of different hexose isomers [20]. Feeding dietary fiber with ⁄ without butyric acid resulted in amelioration which may be a consequence of controlling blood glu- cose levels (Table 1). Diet, in particular, plays a major role in the management of diabetes. Butyric acid, a metabolite of the anaerobic fermentation of dietary fiber is known to act at the level of gene expression and has shown great promise as an antidiabetic and anticancer agent in addition to having various other biological functions [13]. Analysis by GLC to determine the composition of oligosaccharides revealed the presence of galactose, mannose and N-acetylgalactosamine, which is in agree- ment with the literature [17]. Furthermore, elucidation of the fine structure was carried out by MALDI- TOF MS ⁄ MS after labeling with 2AA to aid in increas- ing the sensitivity of detection. MALDI-TOF MS ⁄ MS has been proven to be a sensitive method for determin- ing the structure of oligosaccharide moieties [9]. Sialic acid was not detected in the oligosaccharides because MALDI-TOF MS ⁄ MS was carried out in positive mode, which leads to its decomposition [21]. The oligo- saccharide structures so proposed revealed presence of tri- and tetra-antennary structures. Fujiwara et al. [3] have demonstrated the presence of nine forms of com- 0 100 0 500 1000 1500 2000 2500 3000 3500 Mass 566.26 889.46 1296.35 1701.82 2188.50 2512.26 2836.17 3323.02 % 15001000 2000 2500 3000 3322.14 3500 4000 4500 5000 5500 m/z 1000 2000 3000 A B Intens. [a.u.] Fig. 4. MALDI-TOF spectra of peak 1 (0.2 - M-eluted) from the SFD group (A) and its re- spective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Fig- s 1 and 2. Laminin oligosaccharide changes during diabetes A. P. Kumar et al. 148 FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS plex oligosaccharide chains, which differed in anten- nary and oligo-lactosamine structure, and small amounts of high mannose-type oligosaccharides which are further differentiated from laminin isolated from Engelbreth–Holm–Swarm tumor cells by terminal galactose and sialic acid residues. The oligosaccharide moieties reported in our study contained mannose predominantly followed by galactose. Considerable alteration in the structures of the oligosaccharide moie- ties was observed during diabetes (Table 3). Such structural alterations in oligosaccharide moieties might lead to functional, mechanical and immunological alterations, giving rise to the pathological complications of long-term diabetes. Some of the differences observed in our result with respect to oligosaccharide structures may be due to the yield of a unique subpopulation of laminin oligosaccharides as a result of the different purification techniques employed as well as the source of laminin. For the oligomannose-type structure, the [M + Na + ] adduct was primarily observed. Labeling of oli- gosaccharides with a fluorescent probe like 2AA has been reported to increase the sensitivity of the tech- nique by almost threefold [7]. MALDI-TOF MS ⁄ MS spectra of oligosaccharides of laminin from the SFC group (Figs 2 and 3) showed that, it contains high mannose-type N-glycans with two puta- tive structures, Hex 10 HexNAc 2 (peak 1 at m ⁄ z 2185) and Hex 14 HexNAc 4 (peak 2 at m ⁄ z 3240). Whereas, the MALDI-TOF MS ⁄ MS spectra of oligosaccharides released from laminin from the diabetic group showed a different N-glycan group, Hex 12 HexNAc 6 (peak 1 at m ⁄ z 3322) (Fig. 4) and Hex 14 HexNAc 5 (peak 2 at m ⁄ z 3443) (Fig. 5), which might point to variation in the oligosaccharide profiling during diabetic status. Sub- sequently, the N-glycan structures in the fiber-fed group were Hex 12 HexNAc 3 (peak 1 at m ⁄ z 2713) (Fig. 6) and Hex 12 HexNAc 5 (peak 2 at m ⁄ z 3119) (Fig. 7), and those in the fiber plus butyric acid-treated group were Hex 10 HexNAc 3 (peak 1 at m ⁄ z 2389) (Fig. 8) and Hex 10 HexNAc 4 (peak 2 at m ⁄ z 2592) (Fig. 9). The possible structure of N-glycan of laminin from different experimental groups as analysed by MALDI- TOF MS ⁄ MS spectrum is summarized in Table 3. In this investigation, diabetes results in changes in the oligosaccharides of laminin. In addition, dietary fiber and butyric acid were also able to bring about subtle changes in oligosaccharide structure in laminin during 0 1000 100 0 500 1000 1500 2000 2500 3000 3500 Mass 566.91 1092.83 1620.17 2148.08 2633.24 2956.91 3443.06 % 1500 2000 2500 3000 3500 4000 4500 3443.27 5000 5500 m/z 1000 2000 3000 A B Intens. [a.u.] Fig. 5. MALDI-TOF spectra of peak 2 (0.3 M-eluted) from the SFD group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Figs 1 and 2. A. P. Kumar et al. Laminin oligosaccharide changes during diabetes FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS 149 diabetes. It would be interesting to delineate the poten- tial mechanism by which such changes are brought about. Feeding dietary materials like fatty acid has been shown to influence changes in oligosaccharides in brain microsomes by feeding an n-3-deficient polyunsaturated fatty acid-deficient or -rich diet, thereby affecting learn- ing potential. This led the researchers to conclude that changes may affect membrane functions through changes in membrane surface physical properties and reactivity against serotonin [22]. In conclusion, structural analysis of N-linked oligo- saccharides of laminin using MALDI-TOF MS ⁄ MS suggests that during diabetes the oligosaccharide sequences of laminin are altered, thereby likely imping- ing on its binding to other extracellular components. Feeding dietary fiber and butyric acid were effective in bringing about changes in oligosaccharide moieties. Materials and methods Materials Streptozotocin, PNGase-F, 2AA and Sepharose 4B were from Sigma-Aldrich (St. Louis, MO, USA). Heparin– Sepharose column matrix was procured from Pharmacia Biotech (NJ, USA). Glucose and creatinine estimation kits were from Span Diagnostics (Surat, India). Vitamins, min- erals and guar gum were from HiMedia Laboratories (Mumbai, India). Wheat bran was procured from the local market. All other chemicals and reagents were of analyti- cal grade. Animals, induction of diabetes and diet Diabetes was induced in male Wistar rats weighing 100– 110 g by using streptozotocin (55 mgÆkg )1 body weight in 0.1 m citrate buffer). The study had prior approval from the Institutional Animal Ethics Committee. After 3 days, animals were grouped into SFC and FFC, SFD, FFD and FFD-500 groups. SFC and SFD groups received AIN-76 basal diet, whereas FFC and FFD groups received AIN-76 diet, wherein starch was replaced with 5% wheat bran and 2.5% guar gum. Wheat bran had a total dietary fiber content of 22% (soluble 2%, insoluble 20%) and was rich in arabinose, xylose and glucose. Guar gum, which is a good source of soluble fiber, was galactomannan in nature. Furthermore, the FFD-500 group received butyric acid sup- plementation at 500 mgÆkg )1 body weightÆday )1 in drinking water. Isolation of laminin from kidney cortex Laminin from kidney was isolated according to Paulsson et al. [23]. Kidneys from individual groups were pooled 0 0 500 1000 1500 2000 565.91 1052.03 1579.07 2064.98 2389.14 2713.12 2500 3000 3500 Mass 100 % 1000 1500 2000 2500 3000 2713.48 3500 4000 4500 5000 5500 m/z 1000 2000 3000 A B Intens. [a.u.] Fig. 6. MALDI-TOF spectra of peak 1 (0.2 M-eluted) from the FFD group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Figs 1 and 2. Laminin oligosaccharide changes during diabetes A. P. Kumar et al. 150 FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS before extraction because of the lower amounts of laminin present. Briefly, kidney cortex was separated and homoge- nized with 20 vol. of extraction buffer (0.15 m NaCl in 0.05 m Tris ⁄ HCl) containing protease inhibitors (2 mm phenylmethanesulfonyl fluoride, 2 mm N-ethylmaleimide and 2 mm benzidine ⁄ HCl). The homogenate was centri- fuged at 27 000 g for 15 min. The extraction was repeated twice and supernatant was discarded. The residue left behind was further extracted using extraction buffer con- taining 10 mm EDTA. After stirring for 2 h at 4 °C, the extract was centrifuged at 8000 g for 10 min and the super- natant solution containing laminin was stored at )20 °C until further use. Purification of laminin Purification of laminin was carried out according to Sak- ashita et al. [24]. The method employed initial partial purifi- cation of the crude extract on Sepharose 4B column followed by affinity purification using heparin–Sepharose column chromatography. Partial purification Partial purification of crude laminin was carried out on a Sepharose 4B column. The column matrix and sample to be loaded were initially equilibrated with 10 mm NaCl ⁄ P i (pH 7.4) containing protease inhibitors. Sample ( 15 mg of protein) was loaded onto the column (1 · 35 cm) and 5 mL fractions at a flow rate of 2.5 mLÆmin )1 were col- lected. The eluates were monitored at 280 nm. Putative laminin containing fractions eluting at void volume were pooled, dialyzed and concentrated. Purification by heparin–Sepharose To the pre-equilibrated heparin–Sepharose matrix-contain- ing column (1 · 5 cm), pooled laminin containing fractions ( 3 mg protein) were loaded and eluted at a flow rate of 10 mLÆmin )1 . The column was washed with NaCl ⁄ P i (three times the bed volume) and the bound protein was eluted using NaCl ⁄ P i containing 0.15 m NaCl. The eluates were monitored at 280 nm and protein containing fractions were pooled, dialysed, concentrated and stored at 4 °Cinthe presence of protease inhibitors for further studies. SDS/PAGE of laminin The purity of laminin was ascertained by 3.5% SDS ⁄ PAGE and visualized by silver staining for bands corresponding to 400 and 200 kDa. 0 1000 % 0 100 500 1000 1500 2000 2500 3000 3500 Mass 566.01 1213.97 1903.18 2471.14 3119.12 1500 2000 2500 3000 3119.51 3500 4000 4500 5000 5500 m/z 1000 2000 3000 A B Intens. [a.u.] Fig. 7. MALDI-TOF spectra of peak 2 (0.3 M-eluted) from the FFD group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Figs 1 and 2. A. P. Kumar et al. Laminin oligosaccharide changes during diabetes FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS 151 GLC analysis Laminin (100–200 lg) was hydrolyzed with 2 m HCl at 100 °C for 2 h. The hydrolysate was repeatedly co-distilled with distilled water and reduced using sodium borohydride. Excess sodium borohydride was destroyed by adding 2 m acetic acid and again repeatedly co-distilled with methanol and dried in a dessicator. It was derivatized using acetic anhydride and pyridine (1 : 1, 1 mL) and processed further to remove the salts. The derivatized products were taken in chloroform and analyzed by GLC. The conditions used for the analysis of neutral and amino sugars are as follows: (a) for neutral sugars, OV-225 column; column temp, 200 °C; injection temp: 250 °C; detection temp: 250 °C; N 2 ,40mLÆ min )1 ; (b) for amino sugars, OV-225 column; column temp, 215 °C; injection temp, 250 °C; detection temp, 250 °C; N 2 , 40 mLÆmin )1 . Preparation of glycopeptides Before the digestion, pronase was preincubated with 0.1 m Tris ⁄ HCl (pH 8.0) buffer containing 2 mm CaCl 2 for 1 h at 50 °C. The enzyme solution was added to laminin (100 lg as protein) at an enzyme substrate concentration of 1 : 50 and incubated at 50 °C with stirring. This was followed by the addition of same amount of enzyme at intervals of 24 and 48 h. The digest was placed in a boiling water bath for 5 min to terminate the enzyme activity. Release of N-linked oligosaccharides The glycopeptides obtained from pronase digestion were sub- jected to PNGase-F digestion for the release of oligosaccha- rides. In brief, to glycopeptides, 4 lL of PNGase-F (500 000 UÆmL )1 ) was added and incubated in water bath at 37 °C overnight. To the digest, 3 vol. of cold ethanol was added and allowed to precipitate at room temperature for 5–10 min. The precipitate was centrifuged at 8000 g for 10 min. The supernatant containing oligosaccharides were collected separately and volume reduced to 10 lL with speed- vac concentrator. Derivatization of oligosaccharides with 2AA Oligosaccharides, obtained as above, were subjected to 2AA labeling for structural elucidation [6]. Before labeling, the 2AA solution was prepared by dissolving 30 mg 2AA and 30 mg sodium cyanoborohydride in 100 lL of a solu- tion containing 4% sodium acetate and 2% boric acid in methanol. 1000 100 0 500 1000 1500 2000 2500 3000 3500 Mass 728.01 1093.03 1417.42 1903.11 2389.08 % 0 1000 2000 3000 A B Intens. [a.u.] 1500 2000 2500 3000 2389.21 3500 4000 4500 5000 5500 m/z Fig. 8. MALDI-TOF spectra of peak 1 (0.2 M-eluted) from the FFD-500 group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B). All other details are as in the legends to Figs 1 and 2. Laminin oligosaccharide changes during diabetes A. P. Kumar et al. 152 FEBS Journal 278 (2011) 143–155 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... (1986) Structure of the aspargine-linked sugar chains of laminin Biochim Biophys Acta 883, 112–126 Laminin oligosaccharide changes during diabetes 18 Kim YS (2004) Altered glycosylation of mucin glycoprotein in colonic neoplasia J Cell Biochem 50, 91–96 19 Charonis AS & Tsilibary EC (1992) Structural and functional changes of laminin and type IV collagen after nonenzymatic glycation Diabetes 41, 49–51... (2001) Changes of oligosaccharides of rat brain microsomes depending on dietary fatty acids and learning task J Neurosci Res 63, 185–195 23 Paulsson M & Saladin K (1989) Mouse heart laminin: purification of the native protein and structural comparison with Engelbreth–Holm–Swarm tumor laminin J Biol Chem 264, 18726–18732 24 Sakashita S, Engvall E & Ruoslahti E (1980) Basement membrane glycoprotein laminin. .. scans of samples were recorded over 5000 Da by using a pulse-delay of 90 ns Recorded data were processed by using grams ⁄ 38[2]6 software (v 3.04, Galactic Industries, Salem, NH, USA) Sample preparation for MALDI-TOF-MS analysis Analytical methods Total sugars were estimated by the phenol–sulfuric acid method [25], sialic acid was estimated by Aminoff’s method [26], protein content was estimated by Lowry’s... energy expenditure in mice Diabetes 58, 1509–1517 13 Smith JG, Yokoyama WH & German JB (2000) Butyric acid from the diet: actions at the level of gene expression Crit Rev Food Sci 38, 259–297 14 Chethan Kumar M, Rachappaji KS, Nandini CD, Sambaiah K & Salimath PV (2002) Modulatory effect of butyric acid – a product of dietary fiber fermentation in experimentally induced diabetic rats J Nutr Biochem 13,... emission set 330 and 420 nm, respectively and oligosaccharide-containing fractions were pooled and concentrated by speedvac to reduce the volume to 100 lL Positive-ion mode MALDI-TOF MS analysis of 2AAlabeled oligosaccharides was performed on a Voyager-DE (Perseptive Biosystems, MA, USA) instrument operating at an accelerating voltage of 24 kV (grid voltage 93%, ion guide wire voltage 0.01%) and equipped... Chandrasekaran S & Marvin LT (1990) A biological role of the carbohydrate moieties of laminin J Biol Chem 265, 12553–12562 3 Fujiwara S, Shinkai H, Deutzmann R, Paulsson M & Timpl R (1988) Structure and distribution of N-linked oligosaccharide chains on various domains of mouse tumor laminin Biochem J 252, 453–461 4 Aumailey M & Neil S (1998) The role of laminins in basement membrane function J Anat 193,... pre-equilibrated with NaCl ⁄ Tris After passing the sample, column was washed with twice the bed volume of NaCl ⁄ Tris and the bound oligosaccharides were stepwise eluted with 0.1– 0.5 m of a-methyl d-glucopyranoside in NaCl ⁄ Tris Fractions of 1 mL, were collected at a flow rate of 10 mLÆmin)1 The fractions were checked for oligosaccharides using a fluorescence spectrophotometer with excitation and emission...A P Kumar et al Laminin oligosaccharide changes during diabetes A Intens [a.u.] 3000 2000 2592.71 1000 0 1000 100 1500 2000 2500 3000 3500 m/z 4000 4500 5000 5500 B % 2592.05 Fig 9 MALDI-TOF spectra of peak 2 (0.3 M-eluted) from the FFD-500 group (A) and its respective MALDI-TOF MS ⁄ MS spectra (B) All other details are as in the legends to Figs 1 and 2 890.16 2055.92 1579.05 0 500... 13, 522–527 15 Kumar AP, Chougala M, Nandini CD & Salimath PV (2010) Effect of butyric acid supplementation on serum and renal antioxidant enzyme activities in streptozotocin induced diabetic rats J Food Biochem 34, 15–30 16 Knibbs RN, Perini F & Goldstein IJ (1989) Structure of the major Concanavalin A reactive oligosaccharides of the extracellular matrix component laminin Biochemistry 28, 6379–6392... plate with 2,5-dihydroxybenzoic acid (1 mgÆmL)1 in H2O) at a ratio of 1 : 3 as a matrix MALDI plate was dried under vacuum and used to record the mass spectra Purification of oligosaccharide MALDI-TOF MS 2AA-labeled oligosaccharides were purified into individual oligosaccharide fractions by Concanavalin-A–Sepharose lectin-affinity column chromatography In brief, 1 mL of labeled oligosaccharides was loaded . Structural characterization of N-linked oligosaccharides of laminin from rat kidney: changes during diabetes and modulation by dietary fiber and butyric. sequence of laminin during diabetes which were altered by the feeding of dietary fiber and butyric acid. Abbreviations 2AA, 2-anthranilic acid; FFC, dietary fiber- fed