Background
Stroke is one of the leading causes of death and permanent disability worldwide [
1,
2]. Approximately 80% of stroke is ischemic, whereas 20% is due to primary hemorrhage [
3]. Ischemic stroke (IS) is a multifactorial and complicated disease, resulting from an intricate interplay between environmental and genetic factors [
3]. Cerebral arterial stenosis (CAS) of the major arteries in the brain is one of the most common pathomechanisms for the development of ischemic stroke [
4]. There is a pressing need for better understanding and prevention of CAS. CAS and IS are characterized by a pro-inflammatory state and elevated levels of markers of inflammation, such as C-reactive protein and interleukin-6, which are associated with the risk of CAS and IS [
5‐
8]. Although increasing evidence has indicated that inflammation is important in the progression of CAS to IS [
7,
8], and numerous risk factors have been identified, the underlying mechanisms of IS remain largely unknown.
By attaching to individual proteins, glycans participate in many key biological processes, including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis [
9,
10]. Glycans were also found to be involved in the pathophysiology of some major diseases, such as inflammatory diseases and cancers [
11‐
13]. Glycans have a particularly important role in the immune system, and glycosylation may affect the function of the immune system on multiple levels [
13,
14]. As glycans do not have a direct genetic template, glycan structures are determined by complex dynamic interactions between a number of genetic and environmental factors [
15,
16]. These unique qualities have promoted interest in the role and use of
N-glycans as potential predictors of the development of complex diseases such as IS.
Immunoglobulin G (IgG) plays an important role in the human immune system, and
N-glycans attach to the conserved asparagine 297 in the fragment crystallizable (Fc) region and act as a switch between pro- and anti-inflammatory IgG functionality [
17]. Changes in IgG glycosylation are not only involved in several inflammatory diseases [
18‐
21] but also can be a part of the molecular mechanism leading to the promotion of inflammation [
22‐
24]. The inflammatory role of IgG
N-glycosylation, together with its association with the risk factors of ischemic stroke, such as aging, obesity, dyslipidemia, type 2 diabetes (T2D), and hypertension [
25‐
28], led us to hypothesize that the changes in IgG
N-glycan profiles are involved in the pathogenesis of IS by regulating the inflammatory response.
In this study, we investigated the association of IgG N-glycan profiles with CAS and IS to provide biomarkers for the screening of CAS and IS. In addition, we aimed to determine the association between IgG N-glycome and markers of inflammation, including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), and matrix metalloproteinase-9 (MMP9), to further elucidate the roles of IgG glycosylation on CAS and IS.
Discussion
Previous studies showed that IgG glycosylation is associated with the risk factors of IS, such as aging, obesity, dyslipidemia, T2D, and hypertension [
25‐
28]. In this study, we described the differences of glycosylation of IgG in IS patients and compared these with CAS and the controls. To our knowledge, this study is the first attempt to investigate the association of IS with IgG
N-glycans.
We showed the high levels of GP1 and GP8 and low levels of GP5, GP13, GP14, GP15, GP17, GP18, GP21, and GP23 in IS patients compared with the controls. GP1 and GP8 are agalactosylated and fucosylated glycans, which are significantly higher in IS. In parallel, GP13, GP14, and GP15, which have two galactoses and share the derived trait G2n, are significantly lower in IS. In addition, GP17, GP18, GP21, and GP23 that contain sialic acid are lower in IS. The changes of galactosylation and sialylation for initial glycans are consistent with the results of the association between the derived traits in the IgG glycome and IS. The changes of derived glycans indicate that the high level of bisecting GlcNAc can increase the risk of IS. Although we did not identify the differences of glycans between CAS and controls, the differences of glycans in IS compared with CAS or the controls were identified. The changes of glycans between IS and CAS were less compared to the changes between IS and controls, suggesting that there were some changes of glycosylation in CAS compared with controls. In the progression of IS, the faintly aberrant IgG glycosylation in CAS might play a cascading role in the pathogenesis of IS. Our findings also indicate that the high level of bisecting GlcNAc and the low level of galactosylation and sialylation may increase the risk of IS compared with CAS and controls.
Abundant evidence has shown that the decreasing galactosylation and sialylation and the increasing bisecting GlcNAc are risk factors of many inflammatory and chronic diseases [
18‐
21,
25,
27,
28,
43,
44], which is consistent with our present results (Table
3). As summarized in Table
3, the decreased IgG galactosylation, which was reported in a number of different inflammatory and chronic diseases, might suggest that aberrant glycosylation of IgG is not disease-specific, but a general phenomenon associated with reducing the anti-inflammatory function of circulating IgG. Studies have shown that the absence of sialic acid dramatically changes the physiological role of IgG, converting them from anti-inflammatory to pro-inflammatory agents [
45]. So far, this evidence clearly shows that IgG glycosylation plays a crucial role in modulating the antibody-mediated response and could be a part of the molecular mechanism leading to the promotion of inflammation. In the present study, the association between IgG glycosylation and inflammatory status showed that low levels of galactosylation and sialylation and high levels of bisecting GlcNAc correspond with the state of inflammation.
Table 3
The change of glycans in diseases by UPLC
GP1 | ↑ | – | – | ↑ | ↑ | – | – | – | – | – | – | ↑ |
GP2 | ↑ | – | – | ↑ | ↑ | ↑ | – | – | – | – | ↑ | – |
GP3 | ↑ | – | – | – | – | – | – | – | – | – | – | – |
GP4 | ↑ | ↑ | ↑ | ↑ | – | – | – | ↑ | – | – | ↑ | – |
GP5 | ↑ | – | – | ↑ | – | – | ↓ | – | – | – | ↑ | ↓ |
GP6 | ↑ | ↑ | ↑ | ↑ | – | ↑ | – | ↑ | ↑ | – | ↑ | – |
GP7 | – | – | – | ↑ | – | – | – | – | – | – | – | – |
GP8 | ↓ | – | – | ↓ | – | – | ↑ | – | ↓ | – | – | ↑ |
GP9 | ↓ | ↓ | ↓ | ↓ | – | – | – | – | ↓ | – | – | – |
GP10 | ↓ | – | – | ↑ | – | – | – | – | ↑ | – | – | – |
GP11 | ↓ | – | – | – | – | – | – | – | ↑ | – | ↑ | – |
GP12 | ↓ | – | – | ↑ | – | – | – | ↓ | – | – | – | – |
GP13 | ↓ | – | – | – | – | – | – | ↓ | – | – | – | ↓ |
GP14 | ↓ | ↓ | ↓ | ↓ | – | ↓ | ↑ | ↓ | – | ↓ | ↓ | ↓ |
GP15 | ↓ | – | – | – | – | – | – | ↓ | – | – | – | ↓ |
GP16 | ↑ | – | – | ↓ | – | – | – | – | – | – | – | – |
GP17 | – | – | – | ↑ | – | – | ↓ | – | – | – | – | ↓ |
GP18 | ↓ | ↓ | ↓ | ↓ | – | ↓ | – | ↓ | – | ↓ | ↓ | ↓ |
GP19 | ↓ | – | ↓ | ↑ | – | – | – | – | – | – | – | – |
GP20 | – | – | – | – | – | – | ↓ | – | – | – | ↑ | – |
GP21 | – | – | – | – | – | – | ↓ | – | – | – | ↑ | ↓ |
GP22 | ↓ | – | – | ↑ | ↓ | – | ↓ | – | – | – | – | – |
GP23 | – | – | – | ↓ | – | – | – | – | – | – | – | ↓ |
GP24 | – | – | – | ↑ | ↑ | – | – | – | – | – | – | – |
Galactosylation | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ | ↓ |
Sialylation | ↓ | – | ↓ | ↓ | – | ↓ | ↓ | ↓ | ↓ | – | ↓ | ↓ |
Fucosylation | ↑ | – | – | ↓ | ↑ | – | – | ↓ | ↓ | – | – | – |
Bisecting GlcNAc | ↓ | – | ↑ | ↑ | – | ↑ | – | ↑ | ↑ | – | ↑ | ↑ |
The high levels of pro-inflammatory parameters (CRP, TNF-α, and MMP9) in the IS group show that inflammation might be one of the characteristics of IS. This is supported by the fact that a chronic level of inflammation is a known risk factor for IS [
7]. Therefore, our finding that IS is associated with the inflammatory status of IgG (lower of galactosylation and sialylation and higher of bisecting GlcNAc) might partly explain the inflammation accompanying the development of IS.
The emergence and development of molecular markers opens the “black box” of disease development.
N-glycosylation of human proteins is an essential posttranslational modification, which is closely related to biological function, and may predict the occurrence and development of diseases more accurately [
10,
46]. However, there are many challenges to transfer glycosylation biomarkers into clinical applications. As we have shown (Table
3), the change of glycans (GP5, GP8, GP17, and GP21) in several chronic diseases is not consistent. To select biomarkers for a disease screen, we first chose qualitative biomarkers to determine what conditions can increase the risk of disease. In combination with previous studies (Tables
2 and
3), we found that high level of GP1, GP2, GP4, and GP6 and low level of GP13, GP14, GP15, GP18, and GP23 can increase the risk of disease. Therefore, GP1-2, GP4, GP6, GP13-15, GP18, and GP23 may be developed as clinically useful biomarkers for chronic disease in the future.
In the present study, however, it is worth mentioning GP5 which was incorporated into the two final discriminatory models. In addition, only GP5 was used to discriminate IS from CAS with AUC 0.740. From the results of associations between initial glycans and markers of inflammation, GP5 had a strong negative association with inflammation. Based on these results, low levels of GP5 can increase the risk of IS, which is consistent with the association with Parkinson’s disease but contrary to the association with colorectal cancer, systemic lupus erythematosus, and dyslipidemia [
18,
43]. A possible explanation for this phenomenon could be that IS and Parkinson’s are diseases of the nervous system and have a common pathological basis of cerebral arteriosclerosis [
47‐
49]. In fact, GP5 is not only an agalactosylated glycan but also a high-mannose
N-linked glycan. Studies have shown that high mannose can inhibit the inflammatory effect of macrophages and regulate the immune system to exert anti-inflammatory effects, while decrease in mannose levels can promote inflammatory effects [
50]. In our present study, GP5 may be a special biomarker for the prevention or intervention of IS.
Although the inflammatory role of IgG N-glycosylation associates with the increased risk of ischemic stroke, this novel biomarker needs further validation in multiple-ethnic population and the development of standard operating procedure before the practical utility in screening or diagnosis of this disease. Besides the genome, epigenome, transcriptome, proteome, and metabolome, the glycome would supply new alternative for the screening of the biomarkers. However, there are several common limitations and insufficiencies, which should be recognized. First, the differential glycosylation described above may provide exciting insights into the pathogenesis of ischemic stroke. However, causation is difficult to infer in data from those already diagnosed with the condition, and the observed changes may be consequences rather than causes of the disease. Second, based on the fact that our study is a case-control study, the selection bias cannot be ignored, which could possibly lead to over-estimations of diagnostic accuracy compared with a cross-sectional study or cohort study. In addition, the exclusion criteria with history of medications during the preceding 2 weeks are limited to the controls but do not require the cases, which may trigger the bias in estimation of measuring parameters. Also, only three pro-inflammatory parameters (CRP, TNF-α, and MMP9) were examined to represent inflammatory status, which may induce evaluation bias. Finally, a possible explanation for a failure to demonstrate the differences of glycans between CAS and controls could be that the sample size is too small to identify the differences. Therefore, further cross-sectional studies and cohort studies with large samples are needed for the identification of diagnostic biomarkers for ischemic stroke and a more definite explanation about the relationships between N-glycan structures and ischemic stroke based on our observation.