Alteration of N-Glycan Profiles in Diabetic Retinopathy

Abstract

Purpose: To investigate the alteration of vitreal N-glycans in patients with proliferative diabetic retinopathy (PDR).

Methods: Plasma and vitreous samples were collected from 17 patients (10 females and 7 males) with PDR (PDR group) and 17 nondiabetic patients (8 females and 9 males) with epiretinal membrane (ERM) and idiopathic macular hole (MH) (non–diabetes mellitus [DM] group). Profiles of N-glycans were analyzed by a glycoblotting-based high-throughput protocol that we recently developed. Human retinal microvascular endothelial cells (HRMECs) were cultivated with culture media containing either low glucose (5 mM) or high glucose (25 mM), and expression levels of sialyltransferases were analyzed by real-time PCR and ELISA.

Results: Amount of N-glycans in the vitreous fluid of the PDR group was significantly higher than that of the non-DM group (495.5 ± 37.4 vs. 142.7 ± 30.8 pmol/100 μg protein, P < 0.005), whereas there was no significant difference in the plasma samples between the PDR and the non-DM group. In addition, profile analysis showed that N-glycans with sialic acids increased in the vitreous of the PDR group (328.4 ± 25.8 pmol/100 μg protein) compared to the non-DM group (92.1 ± 21.2 pmol/100 μg protein, P < 0.0005). Expression levels of sialyltransferases ST3GAL1 and ST3GAL4 were upregulated in the HRMECs after high-glucose stimulation. Consistent with the real-time PCR data, high-glucose stimulation elevated the protein levels of ST3GAL1 (117.4 ± 14.9 pg/mg, P < 0.01) and ST3GAL4 (6.1 ± 0.9 pg/mg, P < 0.05) in the HRMECs compared with the cells cultured with low-glucose culture media (ST3GAL1, 64.4 ± 5.8 pg/mg; ST3GAL4, 3.8 ± 0.3 pg/mg).

Conclusions: Our data demonstrate distinct changes in the N-glycan profile and an increase in sialylated N-glycans in eyes with PDR.

Over the past several decades, changes in human behavior and lifestyle have resulted in an acute increase in diabetes worldwide.¹ Since the prevalence of diabetic retinopathy (DR), a severe ocular complication of diabetes, is closely related to the duration of diabetes,² with the growing diabetes epidemic, the world should experience an explosion in the number of patients with DR. However, despite extensive basic research and clinical studies, biochemical mechanisms responsible for the pathogenesis of DR remain elusive. 

Glycans are biopolymers bearing biological information. Since proteins acquire their properties in the presence of glycans on their structure, alteration of glycans is known to vary the function of proteins.³ Therefore, much attention has been paid to the profile changes of N-glycans, which are among the glycans binding to nitrogen (N) in the side chain of asparagine residues on peptide chains, in systemic diseases. For instance, it has been reported that N-glycans are increased in the sera of patients with cancers^4,5 and inflammatory diseases.⁶ Lines of evidence have indicated that structural alterations of N-glycans are related to the pathogenesis of diseases, and thus the profile changes of N-glycans in ocular diseases such as DR are of great interest. However, to date, detailed information about the N-glycan profiles in eyes with DR has not been reported, possibly due to the technical difficulties in the analysis of glycans in minute amounts of samples, for instance, the vitreous fluid. Recently, we established a technique to analyze the glycan profile in human vitreous fluid samples and reported the profile of N-glycans in vitreous obtained from patients with epiretinal membrane (ERM) and macular hole (MH).⁷ 

In this study, we investigated the profile of N-glycans in patients with proliferative diabetic retinopathy (PDR). In addition, we explored the possible mechanism that alters N-glycan formation in ocular tissues under diabetic conditions. 

Materials and Methods

Isolation of Human Plasma and Vitreous Fluid Samples

This study was conducted in accordance with the tenets of the Declaration of Helsinki. After receiving approval from the institutional review board of Hokkaido University Hospital (IRB No. 011-0172), written informed consent was obtained from all patients. Plasma and vitreous samples were collected from 17 patients with PDR (10 females and 7 males, PDR group) and 17 patients with MH or ERM (8 females and 9 males, non–diabetes mellitus [DM] group). All the plasma samples were collected preoperatively, and undiluted vitreous samples were collected at the start of pars plana vitrectomy using the transconjunctival sutureless vitrectomy system (23- or 25-gauge). To avoid contamination of tears, blood, and saline, fluids on the conjunctiva were carefully removed before cutter insertion. Characteristics of the subjects enrolled in this study are shown in Table 1. Previously we reported that there was no significant difference in concentration and composition of N-glycans in the vitreous between MH and ERM or between males and females.⁷ Therefore, in this study we used the data points published earlier⁷ as a control group. No statistically significant difference in age was found between the PDR group (60.1 ± 6.7 years old) and the non-DM group (63.9 ± 4.4 years old). 

Table 1

 

Characteristics of the Patients

 

Glycoblotting of Plasma Samples

Glycoblotting was performed according to the established procedure, as shown previously.⁸ Briefly, plasma samples (10 μL each) were applied to Sweetblot prototype 7 (System Instruments Co., Tokyo, Japan), an automated machine for pretreatment and glycoblotting. After enzymatic cleavage of the proteins, N-glycans were captured on BlotGlyco H beads (Sumitomo Bakelite Co., Tokyo, Japan). Since sialic acids are readily removed or decomposed from peptide chains during ionization in mass spectrometry analysis, we integrated sialic acid protection using the reagent 3-methyl-1-(p-tolyl)triazene (MTT).⁹ This reagent directly methyl esterifies sialic acids trapped on the beads. Consequently, quantitative analysis by mass spectrometry was performed for sialylated N-glycans simultaneously with neutral glycans. Subsequently, the processed glycans were tagged with aminooxy-functionalized peptide reagent (aoWR) and released from the beads, followed by detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS; Ultraflex 3; Bruker Daltonics, Billerica, MA, USA). The intensity of the isotopic peaks of each glycan was normalized to an internal standard (A2 amide glycan) with a known concentration. 

Glycoblotting of Vitreous Samples

Vitreous N-glycans were analyzed with the procedure described in our previous study.⁷ The vitreous samples (200-μL each) were pretreated for enzymatic cleaving and processed in a modified glycoblotting procedure. Based on the structures, glycans were classified into three groups: sialylated N-glycans, high-mannose N-glycans, and others. 

Cell Culture

Human retinal microvascular endothelial cells (HRMECs; Cell Systems Corporation, Kirkland, WA, USA) were cultured in CS-C complete medium (Cell Systems Corporation) containing 5 mM glucose, which corresponds to blood glucose concentration under normal conditions. For high-glucose stimulation, HRMECs were cultured in CS-C complete medium containing 25 mM glucose, which corresponds to blood glucose concentration under hyperglycemic conditions. 

Real-Time PCR

Expression levels of ST3GAL1, ST3GAL4, and ST6GAL1 mRNA were examined by real-time PCR. Total RNA was extracted from the HRMECs using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). Reverse transcription was performed with GoScript Reverse Transcriptase (Promega, Madison, WI, USA) and oligo dT(20) primers following the manufacturer's instructions. TaqMan probes for ST3GAL1, ST3GAL4, and ST6GAL1 were purchased from Life Technologies. Real-time PCR was performed using the GoTaq qPCR Master Mix (Promega), THUNDERBIRD Probe qPCR Mix (TOYOBO, Tokyo, Japan), and StepOnePlus Real-Time PCR System (Life Technologies). The primers used for human GAPDH were 5′-CCTGGCCAAGGTCATCCATG-3′ and 5′-GGAAGGCCATGCCAGTGAGC-3′; GAPDH was used as endogenous control. Threshold cycle (C_T) was determined automatically, and relative change in mRNA expression was calculated using the ΔΔC_T values. All PCR reactions were repeated in triplicate, and the average values were used in the statistical analysis. 

ELISA

Protein levels of ST3GAL1 and ST3GAL4 were measured using ELISA kits for human ST3GAL1 (Cusabio Biotech, Wuhan, China) and ST3GAL4 (Blue Gene, Shanghai, China) according to the manufacturers' protocols, and normalized to total protein (BCA Protein Assay Kit; Thermo Scientific, Rockford, IL, USA). According to the manufacturer's protocol, cells were washed three times with ice-cold PBS and were detached gently by using scrapers. The solutions containing the detached cells and PBS were sonicated four times for 5 seconds each on ice and then centrifuged at 17,700g at 4°C for 10 minutes. The samples were applied to the ELISA kits after validation, and the optical density was determined at 450 and 540 nm using a microplate reader (Sunrise; TECAN, Männedorf, Switzerland). 

Statistical Analysis

All results are expressed as the mean ± SEM as indicated. Statistical analysis was performed using the two-tailed unpaired Student's t-test with Bonferroni correction. Differences between the means were considered statistically significant when the probability values were less than 0.05. 

Results

Increase of Total N-glycans in Vitreous Fluid of Patients With PDR

To determine whether the concentrations of N-glycans are altered in plasma and vitreous of patients with PDR compared to patients without diabetes, we analyzed the spectra of N-glycans in 34 plasma and vitreous fluid samples. In plasma, N-glycan concentration was not statistically different between the non-DM group (817.7 ± 43.0 pmol/100 μg protein, n = 17) and the PDR group (878.2 ± 36.4 pmol/100 μg protein, n = 17, P = 0.29). By contrast, the concentration of N-glycans in vitreous fluid of PDR patients (495.5 ± 37.4 pmol/100 μg protein, n = 17) was significantly higher than in non-DM patients (142.7 ± 30.8 pmol/100 μg protein, n = 17, P < 0.005, Fig. 1). However, when the patients with PDR were divided into two groups based on the levels of glycated hemoglobin (HbA1c), according to the general treatment target 7.0%, there was no statistically significant difference in N-glycan concentration in plasma (882.9 ± 46.6 vs. 874.0 ± 57.6 pmol/100 μg protein, P = 0.91) and vitreous fluid (526.5 ± 62.6 vs. 468.0 ± 45.1 pmol/100 μg protein, P = 0.46) between the groups. Furthermore, no significant difference was found in N-glycan concentration in plasma (857.5 ± 43.8 vs. 892.6 ± 55.3 pmol/100 μg protein, P = 0.65) and vitreous fluid (490.0 ± 53.7 vs. 499.4 ± 53.6 pmol/100 μg protein, P = 0.91) between males and females. 

Figure 1

 

Comparison of N-glycan concentrations between non-DM group and PDR group. Total N-glycan concentration in the vitreous fluid obtained from non-DM and PDR patients. n = 17 each; *P < 0.005.

Alteration of N-glycan Profiles in Vitreous Fluid of Patients With PDR

Next, to investigate the alteration of N-glycan profiles under diabetic conditions, structures of N-glycans were analyzed in plasma (Table 2) and vitreous fluid (Table 3) obtained from patients with PDR and those without diabetes. Thirteen sialylated N-glycans were identified in the vitreous fluid from both groups, and the total concentration of sialylated N-glycans in the vitreous fluid of the PDR group (328.4 ± 25.8 pmol/100 μg protein, n = 17) was significantly higher compared with that of the non-DM group (92.1 ± 21.2 pmol/100 μg protein, n = 17, P < 0.0005, Fig. 2). There was no significant difference in plasma concentration of sialylated N-glycans between the non-DM group (580.3 ± 36.2 pmol/100 μg protein, n = 17) and the PDR group (625.5 ± 25.9 pmol/100 μg protein, n = 17, P = 0.32). Similar to the total N-glycan concentration, when the patients with PDR were divided into two groups based on the levels of HbA1c (7.0%), there was no statistically significant difference in sialylated N-glycan concentration in plasma (632.0 ± 48.0 vs. 619.8 ± 27.2 pmol/100 μg protein, P = 0.83) and vitreous fluid (331.8 ± 49.3 vs. 325.4 ± 25.0 pmol/100 μg protein, P = 0.91) between the groups. 

Table 2

 

N-Glycan Profile in Plasma of Non-DM and PDR

 

Table 3

 

N-Glycan Profile in Vitreous Fluid of Non-DM and PDR

 

Figure 2

 

Alteration of N-glycan profiles in the vitreous fluid of PDR. Concentration of sialylated N-glycans in the vitreous fluid of non-DM and PDR patients. n = 17 each; *P < 0.0005.

As for high-mannose N-glycans, five N-glycans with high-mannose structure were detected in the vitreous fluid obtained from both non-DM and PDR patients, and there was no significant difference in the total concentration of high-mannose N-glycans between non-DM (10.9 ± 2.0 pmol/100 μg protein, n = 17) and PDR (12.4 ± 1.2 pmol/100 μg protein, n = 17, P = 0.54). 

Impact of High-Glucose Stimulation on Sialyltransferases in HRMECs

The current data showed that sialylated N-glycans were exclusively increased in the vitreous fluid of PDR, indicating the possibility that sialylation is induced in eyes with DR; therefore, to investigate the mechanism underlying sialylated N-glycan elevation in the vitreous of PDR, mRNA expression levels of sialyltransferases including ST3GAL1, ST3GAL4, and ST6GAL1 under high-glucose conditions were analyzed using real-time PCR. The expression level of ST3GAL1 mRNA in HRMECs was significantly increased at 6, 24, and 72 hours after maintenance of cultivation with culture media containing 25 mM glucose in comparison with HRMECs cultivated with culture media containing 5 mM glucose (Fig. 3A). Similarly, mRNA expression of ST3GAL4 was significantly increased in 6, 24, and 72 hours in HRMECs under high-glucose conditions (Fig. 3B). By contrast, ST6GAL1 expression showed no upregulation when stimulated with culture media containing 25 mM glucose (Fig. 3C). 

Figure 3

 

Impact of high-glucose stimulation on sialyltransferase expression in HRMECs. Bars represent real-time polymerase chain reaction (PCR) analysis of (A) ST3GAL1, (B) ST3GAL4, and (C) ST6GAL1 at 6, 24, and 72 hours after maintenance in culture media containing 25 mM glucose. n = 5 or 6 each; *P < 0.05.

Concurrent with the real-time PCR data, the protein level of ST3GAL1 (117.4 ± 14.9 pg/mg total protein, n = 5) was increased in the cell lysates of HRMECs cultured under high-glucose conditions (25 mM glucose) compared with those cultured under normal glucose conditions (64.4 ± 5.8 pg/mg total protein, n = 4, P < 0.01, Fig. 4A). Likewise, protein level of ST3GAL4 was significantly increased in the cell lysates of HRMECs when stimulated with 25 mM glucose (6.1 ± 0.9 pg/mg total protein, n = 5) compared with 5 mM glucose (3.8 ± 0.3 pg/mg total protein, n = 4, P < 0.05, Fig. 4B). 

Figure 4

 

Impact of high-glucose stimulation on protein levels of sialyltransferases in HRMECs. Bars indicate average protein levels of (A) ST3GAL1 (n = 4 or 5; *P < 0.01) and (B) ST3GAL4 (n = 4 or 5; *P < 0.05) in the cell lysates of HRMECs 72 hours after stimulation with high glucose (25 mM).

Discussion

In the present study, we demonstrated that the vitreous N-glycan concentration was significantly higher in patients with DR than in those without diabetes; that sialylated N-glycans were increased in the vitreous fluid of patients with DR; and that high-glucose stimulation augmented the sialyltransferases ST3GAL1 and ST3GAL4, but not ST6GAL1. 

Previously, we reported that there was no significant difference of concentration and profile of N-glycans in the vitreous between MH and ERM, or between males and females.⁷ The previous data demonstrated that vitreous fluid samples obtained from patients with MH and ERM may be used as a control group in N-glycan analysis of vitreous samples from eyes with DR, similarly to protein analysis. In this study, compared with the control group, vitreous N-glycan concentration per protein unit was increased in the PDR group. It has been demonstrated that the N-glycan profile is changed in patients with diabetes. For instance, N-glycan structures on proteins such as α1-acid glycoprotein were changed in patients with diabetes.¹⁰ In addition, N-glycan concentration is elevated in tear fluid obtained from diabetic patients.¹¹ Previous data and the current data indicate that N-glycan synthesis undergoes profound changes in the body, including the eyes, with the development of diabetes. 

Next, we sought to explore further details of N-glycan alteration in eyes with DR. Quantitative and profile analysis showed that the concentration of sialylated N-glycans in the vitreous fluid of the PDR group was higher than in the non-DM group, while high-mannose N-glycan concentration in the vitreous was not statistically different between the groups. Since concentrations of high-mannose N-glycans and sialylated N-glycans in plasma were higher in comparison with those in the vitreous, vitreous concentrations of both N-glycans would increase in the case of blood contamination. However, in the current study sialylated N-glycans alone increased in the vitreous of patients with PDR. Hence, it is possible that the increase of vitreous sialylated N-glycans is not simply due to the serum protein influx caused by blood contamination and elevated vascular permeability in eyes with DR. 

There are several putative mechanisms that elevate sialylated N-glycans in DR eyes, for instance, increased synthesis of sialylated N-glycans and suppression of sialylated N-glycan decomposition. Therefore, as a possible mechanism, we sought to determine whether diabetic changes alter the expression of enzymes, sialyltransferases, that mediate the addition of sialic acid to the terminal portions of glycoproteins, and the current data indicate that sialylation is induced in ocular tissues under diabetic conditions. In this study, under high-glucose conditions sialyltransferases ST3GAL1 and ST3GAL4, but not ST6GAL1, were induced in human retinal vascular endothelial cells. Sialyltransferase ST3GAL1 and ST3GAL4 mediate α2,3-sialylation on N-glycans,¹² while ST6GAL1 is involved in the formation of terminal α2,6-sialic acid linkages.^12,13 On N-glycans, sialic acid exists in either α2,3 or α2,6 linkage, and the addition of sialic acid in these linkages is mutually exclusive; that is, the presence of sialic acid at one site in one specific linkage prevents addition of the other.¹⁴ It has been demonstrated that α2,6-sialic acid linkages alter cellular adhesion and invasion on both extracellular matrix and basement membrane components.¹⁴ However, in the current study sialyltransferases ST3GAL1 and ST3GAL4 were induced in the presence of a high glucose concentration, indicating that α2,3-sialylation contributes to the increase of sialylated N-glycans and plays a role in eyes with PDR. 

Little information is available regarding the relevance of sialylation in DR. However, it was previously reported that inflammation upregulated the expression level of ST3GAL1 in mice.¹⁵ In addition, increased α2,3-sialic acid residues on N-glycans are a common feature during neural cell apoptosis.¹⁶ More interestingly, a recent study showed that vascular endothelial growth factor (VEGF) enhances ST3GAL1 expression.¹⁷ The emerging evidence suggests a role of α2,3-sialylation in the pathogenesis of DR. Further studies are expected to elucidate the pathophysiological implications of α2,3-sialylation in DR. 

Recently, it was reported that glucose depletion reduced glycan precursors and decreased sialylation on monoclonal antibody secreted by CHO cells.¹⁸ In addition, the current data showed an increase of sialyltransferases caused by glucose stimulation; it is possible that glucose level is associated with alteration of N-glycan profiles in patients with diabetes. However, due to the glycemic control before surgery, in the present study HbA1c levels in patients with PDR were in a narrow range, and correlation between N-glycan concentration and HbA1c levels could not be analyzed. The relationship between glucose and N-glycan profile remains to be elucidated. 

The present study showed an increase of N-glycans, in particular sialylated N-glycans, in the vitreous of patients with DR compared to patients without diabetes; however, it is likely that complex and diverse causes affect the N-glycan profiles in eyes with DR. For instance, retinal parenchyma and vascular endothelial cells are exposed to an excessive amount of glucose due to elevated glucose levels in the vitreous¹⁹ and hyperglycemia in patients with DR. Therefore, the upregulation of sialyltransferases in cultured retinal vascular endothelial cells under high-glucose conditions, demonstrated in this study, might be related to the increase of sialylated N-glycans in the vitreous fluid of eyes with PDR. However, along with the exacerbation of ischemia caused by retinal capillary nonperfusion in eyes with DR, glucose supply is supposed to be decreased, which presumably causes suppression of sialyltransferase expression in retinal vascular endothelial cells. Therefore, it is conceivable that, apart from the increase in sialylation, other mechanisms elevate the levels of sialylated N-glycans in eyes with PDR, for instance, a decrease in the rate of metabolic breakdown or turnover of the sialylated N-glycans, as well as differences in active transport of these different N-glycans. Moreover, since vascular permeability is increased in eyes with DR, involvement of serum protein infiltration in eyes with DR cannot be completely discounted. Further studies are required to address these unresolved issues. 

In summary, the current study for the first time demonstrates an alteration of glycosylation, in particular sialylation, on the peptide chain in the vitreous of patients with DR. Whereas further studies are still necessary to elucidate the mechanism(s) by which sialylated N-glycans are increased in ocular tissues under diabetic conditions, the current study suggests the importance of qualitative as well as quantitative analysis of proteins in ocular tissue samples. 

Acknowledgments

The authors thank Ikuyo Hirose, Shiho Yoshida, Erdal Tan Ishizuka, and Kazue Okada for their skillful technical assistance on this project. 

Supported by Japan Society for the Promotion of Science KAKENHI Grants 24659754 and 26670749 and JSPS Core-to-Core Program, B. Asia-Africa Science Platforms. The authors alone are responsible for the content and writing of the paper. 

Disclosure: S. Inafuku, None; K. Noda, None; M. Amano, None; T. Ohashi, None; C. Yoshizawa, None; W. Saito, None; M. Murata, None; A. Kanda, None; S.-I. Nishimura, None; S. Ishida, None