N-glycosylation patterns of plasma proteins and immunoglobulin G in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a heterogeneous and complex disease, characterized by lung parenchymal destruction (emphysema), small airways disease (obstructive bronchiolitis), mucociliary dysfunction and chronic airway inflammation [1]. As an important and growing cause of morbidity and mortality, COPD represents a significant burden to the health-care system, with its estimated prevalence of 11.7% (in the adult population) [2]. Moreover, WHO Global Burden of Disease Project predicted COPD to be the third leading cause of death by 2020 [3].

Symptomatology pointing to COPD includes wheeze, dyspnea, chronic cough and sputum production. Establishing a diagnosis of COPD still requires spirometric assessment, regardless of the recent advancements in the COPD management [4]. Spirometry is used to determine the level of airflow limitation, where changes in forced expiratory volume in 1 s (FEV₁) over time used to serve as a measure of disease progression [5]. Yet, considering COPD heterogeneity, patients with similar FEV₁ do not necessarily have the same functional status or underlying pathology, making spirometric assessment alone insufficient for thorough characterization of the individual’s status [6]. Because only a weak correlation between FEV₁, symptoms and impairment of a patient’s health status existed [7], a formal symptomatic assessment was introduced [8]. Also, there was a substantial necessity for measures/biomarkers which would enable reliable patient assessment, management of the treatment and monitoring of the disease progression over shorter periods of time.

To fulfil the aforementioned requirements several potential COPD biomarkers have been investigated recently [9‐14]. Roughly, they could be divided into two subgroups—plasma biomarkers originating from lungs, which can also be denoted as local inflammatory markers, and systemic inflammation-related biomarkers. The most investigated ones from the pneumoproteins group include serum club (Clara) cell protein 16 (CC-16) and surfactant protein D (SP-D) [7]. CC-16 showed associations with accelerated decline in lung function [9], while SP-D associated with an increased risk of exacerbations [10], although observed associations were very weak. Since low-grade systemic inflammation marks COPD, it was hypothesized that certain systemic inflammatory biomarkers would associate with some of the disease features, such as exacerbation frequency or mortality risks. There was a strong evidence of an association between fibrinogen and the presence of COPD, frequency of exacerbations and mortality [14]. Thereby, fibrinogen showed association with disease severity but was not able to predict lung function decline. These findings eventually led to the FDA qualification of plasma fibrinogen as a prognostic or an enrichment factor, in addition to standard inclusion/exclusion criteria, in COPD clinical trials with endpoints of COPD exacerbation and/or all-cause mortality [15]. There are also other promising plasma biomarkers for COPD, such as C-reactive protein (CRP) [16] and serum amyloid A protein (SAA) [17], which are acute phase proteins as well. Taking into consideration the importance of inflammatory component of the COPD, several studies were conducted focusing on several inflammatory biomarkers simultaneously. One study monitored individual’s inflammome, i.e. six inflammatory biomarkers [white blood cells count, CRP, interleukin 6 (IL-6), IL-8, fibrinogen and tumor necrosis factor-α (TNF-α)], observing that persistently inflamed subjects had significantly increased mortality and exacerbation frequency compared to non-inflamed ones [18]. Another study showed that simultaneously elevated levels of C-reactive protein (CRP), fibrinogen and leukocyte count were associated with increased risk of exacerbations, even in individuals with milder COPD and in those without previous exacerbations [11].

The inflammatory response, which clearly plays a significant role in COPD pathophysiology, requires fine tuning of many cellular and extracellular processes, such as complement activation, production of cytokines, release of endothelin and the expression of adhesion molecules on leukocytes and the endothelium. The complex arrangement and highly precise regulation of these molecular mechanisms is, among other, dependent on proper N-glycosylation of involved molecules [19]. N-glycosylation is a co- and posttranslational modification characterized by enzymatic attachment of complex sugar moieties (glycans) to the protein. Correct glycosylation is essential for protein stability and can modulate its function [20]. Previously, it was shown that inflammation associates with disruption of N-glycan processing, resulting in aberrant protein glycosylation [21]. Also, N-glycans have demonstrated to be very sensitive markers of various disease states [22]. Furthermore, all promising COPD biomarkers, including fibrinogen, are in fact glycoproteins, showing that glycans are involved in nearly all (patho)physiological processes. Glycosylation also modulates function of immunoglobulin G (IgG), a potent and versatile effector member of the immune system. Differential N-glycosylation of its fragment crystallizable (Fc) affects IgG effector functions through modified binding affinity to the Fc-receptors (FcγRs), enabling its ability to act as a pro- or anti-inflammatory agent [23]. Therefore, it is also worth exploring IgG glycosylation changes in any disease with the inflammatory component, including COPD. To summarize, glycans could offer a new perspective in the search for COPD biomarkers. Therefore, we aimed to examine the individual variation of plasma protein and IgG glycosylation profiles in subjects with COPD, since this individual variation of N-glycosylation in COPD has, to date, never been investigated. Additionally, we aimed to examine glycan changes associated with the disease severity and to explore whether glycan changes show potential as markers of COPD progression. Finally, since smoking represents a major risk factor in COPD aetiology, our goal was and to address the effects of smoking on plasma protein and IgG glycosylation.

To the best of our knowledge, this study is the first one to address individual variation of plasma protein and IgG N-glycosylation in chronic obstructive pulmonary disease. Plasma protein glycosylation exhibited several extensive changes, which were more pronounced than the changes occurring in the IgG glycome. We observed that subjects with COPD show a decrease in low branched glycoforms (mono- and biantennary glycans) and conversely, an increase in more complex glycan structures. Also, we revealed a significant reduction in plasma monogalactosylated speciesand this change replicated for IgG glycome as well. Increasing complexity of the plasma glycome in COPD is driven mainly through significant augmentation of relative abundance of tetragalactosylated, tri- and tetrasialylated, and antennary fucosylated glycans. Similar changes in plasma glycome have been marked in several other conditions with inflammatory component, such as type 2 diabetes [27] or low back pain [28], as well as in acute systemic inflammation [21]. The common denominator of all these conditions is the increased branching of the glycans, driven mostly by increase in galactosylated and sialylated species, which the aforementioned studies reported. However, these studies did not monitor the levels of antennary fucosylation, which is one of the glycosylation features with the most pronounced change in COPD patients (next to mono- and tetragalactosylation). Glycan structures bearing antennary fucose can participate in forming of sialyl Lewis-X structures, which have a very important role in the initiation of inflammation [29, 30]. Hence, even according to the pattern of glycosylation changes of the plasma proteins, the inflammatory component has an important role in pathophysiology of COPD. As already stated, COPD is characterized by local inflammatory response in the lungs. Although the disease primarily affects the lungs, it can also produce a substantial systemic response, which is here clearly mirrored through the plasma glycome changes. However, a recent study which examined the associations of COPD with the inflammome (systemic inflammatory network pattern) has demonstrated that only 15% of COPD patients exhibit signs of systemic inflammation, and, moreover, 30% of them did not have any abnormal biomarker of systemic inflammation [18]. This could indicate that the plasma N-glycans have a greater sensitivity to the restricted, localized, chronic inflammation than the most of the commonly used serum/plasma biomarkers of inflammation. Furthermore, this could be potentially exploited to monitor the disease progression or patient’s status via plasma glycome alterations. One major drawback we here need to acknowledge, is the fact that we cannot differentiate if the observed plasma glycome changes are in fact the consequence of variation in glycosylation of individual proteins or just the result of their concentration variation. However, we presume that the total plasma protein N-glycome reflects an overall trend of glycosylation changes, exhibiting both changes in glycosylation of individual proteins as well as changes in their plasma concentration, resulting with enhanced sensitivity to the pathophysiological events.

The most prominent change of IgG N-glycome associating with COPD is a decrease in monogalactosylation. In general, this alteration in IgG glycosylation is thought to decrease its immunosuppressive ability, by enabling recognition and binding of agalactosylated IgG by mannose-binding lectin, and sequential activation of the lectin complement pathway [31]. Decrease in galactosylated glycoforms of IgG was previously reported in a different autoimmune and inflammatory diseases [32, 33], therefore, cannot be considered as a disease-specific marker. Recently, there have been increasing indications of an antigen-specific and, possibly, autoimmune response in COPD. Namely, IgG autoantibodies with avidity for pulmonary epithelium (i.e. HEp-2 epithelial cells, bronchial epithelial cells and endothelial cells) and the potential to mediate cytotoxicity, seem to be prevalent in patients with COPD [34]. Therefore, monitoring IgG glycosylation changes shows prospect in observing the early signs of pathophysiological processes in at-risk subjects.

According to the GOLD initiative definition, COPD is a preventable and treatable disease characterized by persistent respiratory symptoms and airflow limitation [4]. Essentially, there are tools, including particular biomarkers, which can aid in monitoring the patient’s status and disease progression, still, they all show certain limitations. For instance, one of the most extensively used spirometric parameters, FEV₁, is poorly related to other clinically relevant symptoms (7) and requires a substantial length of observation time for disease progression monitoring (6). For that reason, we examined glycan changes in different disease stages. From our results it is notable that plasma glycan changes reflect the progression of the disease, therefore, could be potentially used to stratify patients according to the disease severity or to monitor the disease progression. The newer GOLD ABCD assessment tool had also put an accent onto patient’s clinical symptoms and exacerbation frequency. In the meanwhile, the prevention of the occurrence of exacerbations became one of the major therapeutic goals in COPD [4], since they are a substantial burden for both patient and the health care system. Exacerbations essentially represent an increase in the inflammation that is present in the stable state, being induced by both infectious and non-infectious causes [35‐37]. In some patients, no cause is identified, and presently there are no reliable biomarkers which would predict them [35]. Herein we examined whether glycans have a value in predicting the frequency of exacerbations and observed that monogalactosylated and asialylated plasma glycoforms significantly decreased with the increase of exacerbation frequency. Although glycan changes show potential, their performance was not so powerful to enable a confident prediction of a single annual exacerbation event. However, a recent study [38] has demonstrated that lower levels of IgG1 or IgG2 subclasses resulted in increased risk of exacerbations, where approximately 20% of COPD patients had one or more IgG subclass deficiencies. It would be interesting to analyse subclass-specific glycome in COPD patients to check for associations between glycome composition and exacerbation frequency. Hence, further research would be needed to explore the possibilities of glycan utilisation in this regard.

The last aim of our study was to examine effects of smoking on relative abundances of different glycoforms. The fact that smoking induces changes in glycosylation pattern of various glycoproteins is well known from the previous studies [39, 40]. Herein, smoking resulted in significant increase of IgG glycoforms with bisecting GlcNAc and decrease in core fucosylated glycans, which is consistent with the results from the previous studies [39, 41]. Both increased level of bisecting GlcNAc and decreased level of core fucosylation of IgG glycans coincides with the increased proinflammatory potential of the IgG [42, 43], proving that smoking leads to inflammation [44, 45]. Similarly, the changes in plasma glycome, occurring as a consequence of smoking, also showed inflammation-like pattern—the increase in high branched structures, highly sialylated and antennary fucosylated glycans. These findings somewhat coincide with the previous studies [39, 40], yet, some of them are novel, so they would require further investigation and replication. However, they could be revealing some novel associations, which could help to elucidate the exact mechanisms of studied pathophysiological changes.

Up to date there was no extensive research conducted which would be focused on investigating the alterations of plasma protein or IgG N-glycans in COPD. Ever-growing evidence of the N-glycosylation importance is demonstrating that the glycan parts of glycoprotein are as essential as the protein itself, since they are modifying its structure, properties and function [20]. This clearly suggests that the glycans should not be neglected when investigating underlying molecular mechanisms of any studied condition. Also, the possibilities should be explored whether glycans could be useful as predictive or stratifying tools, since they do show the great sensitivity of change in response to the various genetic and environmental influences. This study demonstrated that glycans are able to differentiate COPD patients from the healthy individuals and can distinguish patients in the different stages of the disease, which could potentially be exploited to monitor the disease progression, patient’s status, and even response to treatment. Moreover, glycan profiles showed to be affected by smoking, which is the major risk factor for COPD development, demonstrating that they could help in detecting pathophysiological changes at their earliest. However, due to the cross-sectional design of our study we cannot speculate on causality of those glycan changes, nor can we monitor long-term outcomes. In conclusion, this study provided new insights into glycan changes occurring in COPD and indicated new prospects in the search for markers of COPD progression and severity.