Introduction

Glycans are involved in virtually all biological processes, thus influencing the development, growth and functioning of cells and organisms. Glycans have both structural and modulatory roles in the recognition of both self and non-self molecules. Glycosylation is critical for proteins to assume their native conformation and for properties such as solubility, antigenicity and half-life. Moreover, glycosylation influences the function of many proteins. About half of our body proteins (and nearly all cell surface and secreted proteins) are glycosylated (Apweiler et al 1999). Glycosylation has a key role in all interactions between cells and their environment (e.g. extracellular matrix and serum molecules), and since the immune response is based on innumerable contacts between cells and molecules, glycans play an essential role in the immune interactions.

This literature review addresses the relationship between glycosylation and the immune system, and the immunological consequences of human glycosylation defects, known as congenital disorders of glycosylation (CDG).

Glycosylation at a glance

The two main types of protein glycosylation are N-glycosylation and O-glycosylation. N-glycosylation is the most common covalent modification of proteins in human cells (Stanley et al 2009). It involves an N-glycosidic bound that links the nitrogen of an amide group of an asparagine (Asn) residue to N-acetylglucosamine (GlcNAc) of a glycan. The involved Asn has to be part of the consensus aminoacid sequence Asn-X-Thr (where X stands for any aminoacid, except proline).

The N-glycosylation involves many different enzymes and transporters in the cytosol, the endoplasmic reticulum (ER) and the Golgi. The first step takes place on the cytoplasmic side of the ER, and it is initiated by the addition of a GlcNAc-1-phosphate to dolichol-phosphate, a membrane bound poly-isoprenol lipid. This structure is extended by the addition of a GlcNAc and five mannose (Man) residues. The Man5GlcNAc2-PP-dolichol is then enzymatically flipped to the luminal side of the ER membrane. Next, four mannoses and three glucoses are added and the 14 monosaccharide-glycan is transferred en bloc from dolichol to the growing polypeptide, due to the action of the oligosaccharyltransferase (OST) complex. After this assembly phase in the ER, the glycan is remodelled partly in the ER and in the Golgi. First, three glucose and up to six mannose residues may be removed by the action of glycosidases (Moremen et al 1994), leaving the protein-bound pentasaccharide “core”, Man3GlcNAc2. This core structure is further extended with different sugars, namely GlcNAc, and subsequently galactose, sialic acid and fucose.

O-glycosylation is a covalent modification that occurs on the hydroxyl group of serine or threonine residues, in humans often via an N-acetylgalactosamine (GalNAc). There is a great variability in O-glycans, according to the involved sugars and the bond-type. Contrary to what occurs in N-glycosylation, O-glycosylation is mostly initiated in the lumen of the ER and the assembly phase is not followed by a remodelling phase.

The immune system needs glycosylation

The mechanisms to provide immune protection can be divided into innate and adaptive immune responses that work together to eliminate the pathogens. The innate immune response represents the first defense, and is mediated by different cell types, as monocytes, macrophages, natural killer (NK) cells, neutrophils and antigen presenting cells (APC), such as dendritic cells (DC). Cytokines produced by cells of the innate immune system mediate the strength of a non-specific and inflammatory response.

The adaptive immune response includes B and T cells whose receptors are highly specific to the particular pathogen that engages them. This type of immune response provides immunological memory, thus long-lasting protection. B cells can differentiate into plasma cells, which secrete antibodies. This leads to pathogen elimination due to complement activation and engagement of antibody receptors on immune effector cells. The T cells only recognize peptide epitopes presented in the context of major histocompatibility complex (MHC) molecules. Cytotoxic T cells (Tc, CD8+) have the ability to kill cells infected with virus or intracellular bacteria or malignant cells by recognizing epitopes presented through MHC-I. Helper T cells (Th, CD4+) secrete relevant cytokines and recognize epitopes presented through MHC-II. Depending on the stimuli provided by APCs, Th cells differentiate into subsets, mainly defined by the set of cytokines produced: Th1 (IFN-γ-producing Th), key to the induction of anti-tumor cellular immune responses; Th2 (IL-4-producing Th), which support humoral immune responses; and FoxP3+ regulatory T (Treg) cells, regulating immune responses. The Th17 (IL-17-producing Th) is another subset involved in anti-microbial and inflammatory responses.

Interestingly, all these immunological processes depend at some point on glycans. This is the subject of the relatively understudied field of glycoimmunology. Many of the receptors expressed by immune cells are lectins, i.e. proteins that recognize glycans, including: (i) C-type lectins, that function mostly as receptors for pathogen-associated molecular patterns (PAMPs), participating in the sensing and internalization of pathogens. Some C-type lectins are soluble, such as the collectin subfamily, that activate complement and function as opsonins, facilitating phagocytosis. Selectins are one particular group of C-type lectins, which regulate interaction of leukocytes in the blood with endothelium thus allowing the extravasation of leukocytes from blood into inflamed/infected tissues; (ii) siglecs, that recognize glycans bearing the sugar sialic acid and are involved in the regulation of cellular interactions and signaling; (iii) galectins, which are soluble lectins specific for β-galactoside sugars, that are present both intra- and extracellularly and act as scaffolding adaptor proteins to aggregate other cell surface molecules, thus regulating a variety of downstream signaling mechanisms.

Besides lectins, other immune-related receptors may also recognize glycans or glycosylated epitopes, such as the Toll like receptors (TLR), T cell receptors (TCR) and antibodies. Glycans can also be displayed by APCs through CD1 molecules, which present glycolipid antigens to be recognized by natural killer T (NKT) cells (Barral and Brenner 2007) or MHC-I in case of truncated O-glycans (Jensen et al 1996).

Interestingly, it has now become evident that the diversity of glycans that are expressed at the cell surface of immune cells is essential for immune cell development and function (Crespo et al 2013; Priatel et al 2000; Stanley and Guidos 2009; Baum and Crocker 2009). An example is the organ specific homing of leukocytes which is dependent on their glycan recognition by endothelial selectins (Sperandio et al 2009; Carlow et al 2009). The relevance of cell surface glycans is also illustrated by the large diversity of glycans that decorate specific immunological receptors. This is the case of antibodies, whose capacity to be recognized by Fc receptors expressed on effector cells, and subsequent effector function is dramatically influenced by the glycan content of the Fc region (Anthony and Ravetch 2010).

The terminal monosaccharides on membrane and extracellular proteins are often sialic acids, which are thus on the frontline of interaction in leukocyte communication and overall immune response (Crespo et al 2013). Sialic acid helps masking ligands expressed by host cells from pathogen recognition. Sialic acid on host cells prevents autoimmune responses, by inhibiting complement deposition. During acute phase inflammation, the amount of sialic acid on cell surface is increased, as a consequence of the increase in soluble and circulating sialyltransferase isoforms. This hypersialylation seems to protect cells against pathogens, helping the immune system distinguish ‘self’ from ‘non-self’ antigens and maintain immune response homeostasis (Jamieson et al 1993; Nasirikenari et al 2006). Sialic acids or sialic acid-containing molecules function as recognizable patterns for a number of receptors that modulate the immune response such as siglecs and selectins (Varki and Gagneux 2012). Thus alterations in sialic acid content or glycans decorated by sialic acids play a significant role in the immune response.

CDG and immunological phenotypes

Congenital disorders of glycosylation (CDG) are a group of rare, genetic diseases due to defects in the glycosylation, an essential and complex posttranslational modification of proteins and lipids (Freeze et al 2009; Jaeken et al 2009). More than 85 CDG have been reported. The most common CDG is PMM2-CDG, which was first reported in 1980 (Jaeken et al 1980). Its biochemical basis was identified in 1995 (Van Schaftingen and Jaeken 1995) and the molecular basis in 1997 (Matthijs et al 1997).

Since glycans play important roles in all organs and tissues, CDG are mostly multi-organ diseases. In the majority of CDG the nervous system is involved. This leads to developmental disability, hypotonia, hyporeflexia and ataxia. Other organs can be affected: the liver (Kjaergaard et al 2001), the skeleton (Coman et al 2008), the skin (Rymen and Jaeken 2014), the heart (Jaeken et al 1997), the gonads, the endocrine system, the immune system (Lyons et al 2015) among others.

Biochemical serum abnormalities include altered glycoforms of glycoproteins, which are identified using isoelectric focusing (IEF) to detect hyposialylation/hypoglycosylation of transferrin. It also includes decreased thyroxin-binding globulin, coagulation factors, haptoglobin, ceruloplasmin, and increased serum arylsulphatase A and other lysosomal enzymes and other secondary disturbances, such as increased serum transaminases, and decreased cholesterol and iron (Goreta et al 2012; Scott et al 2014). Since CDG have only a few specific symptoms, they are often misdiagnosed (de Lonlay et al 2001).

The present literature review is based on a search of the Medline database, using PubMed as the search engine. Table 1 lists the keywords used. Table 2 compiles published clinical data, in particular the results for immune cells and molecules associated with immune functions. We divided CDG into two main groups (depicted in Fig. 1, according to their cellular location): CDG with predominant or major involvement of the immune system, where all or a great majority of the reported patients showed important immunological deficiencies, and CDG with minor or variable involvement of the immune system. Figure 1 illustrates the different glycosylation steps that are affected in CDG with involvement of the immune system. Interestingly, CDG which have an extensive involvement of the immune system seem to have a trend towards mutations affecting Golgi-resident enzymes, as depicted in Fig. 1. Importantly, no efficient therapy is known for the CDG here under consideration. For a few among them, a limited treatment is available as indicated.

Table 1 Keywords used in the present review
Table 2 Immunological data from CDG patients
Fig. 1
figure 1

Glycosylation of proteins is performed by a set of enzymes located either on the cytosol or on special organelles such as the endoplasmic reticulum or the Golgi. CDG are caused by mutations affecting specific proteins of the glycosylation pathway. These enzymes are associated with CDG with major (red rectangle) or minor (violet rectangle) involvement of the immune system, as described in this work. CDG with major immunological involvement are PGM3-CDG, ALG12-CDG, MAGT1-CDG, MOGS-CDG and SLC35C1-CDG. CDG with minor involvement of the immune system are PMM2-CDG, ALG1-CDG, PIGY-CDG, DK1-CDG, MAN1B-CDG, MGAT2-CDG and COG6-CDG

CDG with major involvement of the immune system

ALG12-CDG

Nine patients have been reported with this CDG (MIM:607143) due to deficient dolichyl-P-Man:Man7GlcNAc2-PP-dolichol α1,6-mannosyltransferase (EC:2.4.1.260), the enzyme responsible for adding the eighth mannose residue on the Man7GlcNAc2-PP-dolichol. They present variable dysmorphic and neurological symptoms. In five patients there was a higher or relatively high frequency of common infections (ear, nose throat), pneumonia and/or lethal sepsis (Chantret et al 2002; Eklund et al 2005; Grubenmann et al 2002; Kranz et al 2007a, b). This tendency for recurrent infections is probably related to significantly decreased serum IgG levels, measured in four of these five patients. In a few of these patients there was also a decrease of IgA and IgM (Grubenmann et al 2002; Kranz et al 2007a, b). The sibling of one of the patients died at a very early age (67 days) before infections might have developed (Kranz et al 2007a, b; Murali et al 2014) and also showed low serum IgG levels. Other immunological tests were only performed in the sibling that died from infection (Table 2), revealing decreased absolute B cell count, absent antibody response to diphtheria, tetanus and Hemophilus influenzae immunization, but normal T cell markers and mitogen testing. The patient was treated with scheduled gamma globulin infusions, but died before reaching 2 years of age, due to overwhelming sepsis (Kranz et al 2007a, b). Interestingly, in another patient with ALG12-CDG, no infections are mentioned (Thiel et al 2002) and two other patients without infectious diathesis showed normal serum IgG levels (Di Rocco et al 2005). Thus, the majority of the ALG12-CDG patients experience severe infections, associated with hypogammaglobinemia and B cell dysfunction. This may be due to deficient N-glycosylation of immunoglobulins, which is necessary for their effector functions such as complement binding and antibody-dependent cell cytotoxicity and pathogen phagocytosis (Anthony and Ravetch 2010).

MAGT1-CDG

The magnesium transporter 1 (MAGT1) deficiency causes XMEN disease (X-linked immunodeficiency with magnesium defect and EBV infection and neoplasia; MIM:300853), a mild form of primary combined immune deficiency (Li et al 2011), reported in seven male patients. MAGT1 (EC:3.6.3.2), also known as IAP (implantation-associated protein), has been reported to have two, seemingly totally different functions: it selectively transports Mg2+ across the plasma membrane, and on the other hand it is a subunit of the OST complex, which catalyzes the transfer of the oligosaccharide from dolichol to proteins in the N-glycosylation pathway in the ER. The major clinical features of XMEN patients are a persistent elevation of EBV viral load with an associated increased susceptibility to EBV-driven B cell lymphomas/lymphoproliferative disorders, dysgammaglobulinemia and CD4 lymphopenia. These patients often show splenomegaly. Moreover, they have an increased susceptibility to mild viral sinus, ear, lung and other infections. An initial report on intellectual disability in MAGT1 deficiency (Molinari et al 2008) was not supported since the published genetic variant was subsequently shown in patients with no intellectual disability (Piton et al 2013).

MAGT1 is a critical regulator of the intracellular Mg2+ that exists in the free (ionized) state. In normal T cells, stimulation of the TCR triggers a transient Mg2+ flux through MAGT1. This is also required to maintain the expression of NKG2D, a C-type lectin expressed by NK and some T cells, and the normal cytolytic function of these cells. Indeed, XMEN patients show impaired T cell activation and decreased cytolytic function of NK and CD8+ T cells. These two defects lead to failure to clear EBV infection and to increased EBV-associated malignancy. B cells have not been shown to be directly affected. Since MAGT1 is a subunit of the OST complex, one would expect that this disorder is associated with an N-glycosylation defect but this has still to be investigated.

As to treatment, Mg2+ supplementation safely increases the basal intracellular Mg2+ pool in XMEN lymphocytes and NK cells, increasing NKG2D expression and decreasing EBV viremia. Therefore, clinical trials with Mg2+ supplementation in patients are underway. XMEN has thus revealed a previously unknown function of free intracellular Mg2+ in the immune function.

MOGS-CDG

MOGS-CDG (MIM:606056) is due to defective mannosyl-oligosaccharide glycosidase (MOGS), also known as ER glucosidase I (EC:3.2.1.106), which removes the distal α1,2-linked glucose from the Glc3Man9GlcNAc2 glycan after its transfer to the nascent polypeptide. The serum transferrin IEF test is normal in this disorder and the diagnosis relies on the detection of the Glc(α1-2)Glc(α1-3)Glc(α1-3)Man tetrasaccharide in the urine. It was first reported in a patient, born at 36 weeks of gestation, with severe generalized hypotonia, epilepsy and dysmorphism, who died at the age of 74 days (De Praeter et al 2000). This patient had normal serum immunoglobulin levels, except IgA, which was undetectably low.

Later, Sadat and colleagues reported an immunological phenotype in siblings of 6 and 11 years old. These patients showed, besides dysmorphism and neurological involvement, severe hypogammaglobulinemia with, paradoxically, an increased resistance to particular viral infections (Sadat et al 2014). Patients had normal or increased levels of B cells, but a reduced circulating immunoglobulin half-life (at least of IgG), which explains the hypogammaglobulinemia. The causative mechanism for the hypogammaglobulinemia remains uncertain, however altered N-glycosylation of immunoglobulin may alter binding to certain Fc receptors, such as the neonatal Fc receptor, a process controlling immunoglobulin half-life (Hayes et al 2014). Interestingly, in spite of the hypogammaglobulinemia there were no frequent or severe bacterial infections, for unclear reasons. Notwithstanding, patients developed an adequate response to vaccination with diphtheria-tetanus-acellular pertussis, conjugated Haemophilus influenzae type B, hepatitis B vaccines and the 23-valent pneumococcal polysaccharide vaccine. However, antibody titers were negative or equivocal after vaccination with live viral vaccines such as measles, mumps, rubella and varicella. Patients’ cells, when tested for susceptibility to various viral infections, showed to be resistant to infections with glycosylation-dependent enveloped viruses, such as HIV and influenza viruses. Such viruses, to be able to enter and egress target cell, depend on glycoproteins synthesized by infected host cells, which are probably absent in MOGS-CDG patients. The measles, mumps, rubella and varicella virus are also glycosylation-dependend enveloped viruses. Thus these studies strongly suggested that the N-glycosylation defect in MOGS-CDG patients is responsible for reduced susceptibility to infection by glycosylated enveloped viruses. In line with these findings, it was shown that these siblings had no altered susceptibility to adenovirus or poliovirus 1, which are non-enveloped viruses, or to vaccinia virus, which is an enveloped virus that does not depend on glycosylation for cell entry. Furthermore, it is suggested that ER glucosidases are valuable targets for antiviral agents against enveloped viruses (Chang et al 2015).

SLC35C1-CDG

SLC35C1-CDG is also known as leukocyte adhesion deficiency, type II (LAD2) (MIM:266265). The defective protein is SLC35C1, a GDP-fucose transporter, which is involved in the transfer of GDP-fucose from the cytoplasm (where it is synthesized) to the Golgi lumen (where it is used to fucosylate glycoconjugates).

In most SLC35C1-CDG patients, the phenotype is characterized by a short stature, dysmorphism, neurological involvement, as well as recurrent bacterial infections associated with a persistent, unusually high neutrophil count. Infections are local (sinus, oral mucosa, ears, lungs, intestine) or generalized (septicemia), and tend to decrease after the first years. The fucose deficiency leads to an absent or diminished ability to generate fucosylated glycans and it is not yet clear how this causes the neurological symptoms. Yet, the immunological phenotype is understandable, since the fucosylated glycans include the selectin ligand sialyl Lewis X (sLex), which is required for leukocyte migration and homing (Silva et al 2012). Bacterial infection requires leukocyte recruitment that starts with tethering and rolling interactions between granulocytes and target tissue endothelium, that are principally mediated by the selectins. Hence, in SLC35C1-CDG granulocytes cannot efficiently migrate to sites of infection, which is manifested by marked leukocytosis, an inability to generate pus, and recurrent bacterial infections. Interestingly, these patients also show a lack or decrease of the fucosylated Lewis blood group and ABO antigens resulting mostly in the rare Bombay blood group (Dauber et al 2014; Etzioni et al 1992; Frydman et al 1992; Hidalgo et al 2003; Lübke et al 1999; Marquardt et al 1999a, b).

Oral fucose supplementation is used to treat SLC35C1-CDG patients but, depending on the nature of the mutation, only some patients have shown a satisfactory response. Fucose, as expected, improves the neutrophil count and decreases recurrent infections (Etzioni and Tonetti 2000; Hidalgo et al 2003; Lühn et al 2001; Marquardt et al 1999a, b).

PGM3-CDG

This CDG (MIM:615816) is caused by deficient phosphoglucomutase 3 (or phosphoacetylglucosamine mutase 1; EC:5.4.2.3) that catalyzes the conversion of GlcNAc-6-P into GlcNAc-1-P, the last step in the synthesis of UDP-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. Twenty-two patients belonging to ten families have been reported (Lundin et al 2015; Sassi et al 2014; Stray-Pedersen et al 2014; Zhang et al 2014). Besides a variable neurological, dysmorphic and skeletal involvement, all patients display an important immunological phenotype characterized by recurrent bacterial infections (particularly Staphylococcus aureus), viral as well as fungal infections, allergy, and atopy. Various combinations of otitis media, upper respiratory infections, bronchitis, pneumonia, bronchiectasis (sometimes leading to respiratory failure), pericarditis, arthritis, skin infections, (lethal) sepsis, influenza, severe varicella or EBV infections have been reported. Eczema/atopic dermatitis was present in 90 % of patients. Allergic manifestations included asthma, allergic rhinitis, and food and drug allergy. Autoimmune diseases were diagnosed in a few patients as cutaneous leukocytoclastic vasculitis or membranoproliferative glomerulonephritis. The serum transferrin IEF test is normal in these patients and thus cannot be used for diagnosis. Two patients, identical twin boys, developed Hodgkin lymphoma and were successfully treated with chemotherapy (Zhang et al 2014). Bone marrow failure occurred in a brother and sister. These patients were treated with hematopoietic stem cell transplantation, with successful neutrophil and T and B cell correction (Stray-Pedersen et al 2014).

Laboratory findings in most patients revealed abnormalities of immune cells and immunoglobulins, which explain the immunopathology. This includes neutropenia, lymphopenia, eosinophilia, an increased CD4/CD8 ratio, decreased T cell and B cell numbers and decreased NK cells reported (Lundin et al 2015; Stray-Pedersen et al 2014; Zhang et al 2014). Granulocytes showed normal chemotaxis and surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins, which regulate leukocyte adhesion, polarization and motility. Serum levels of IgE were normal to highly increased, and levels of IgA, IgG and IgM were low, normal or increased. Serum IgE glycosylation of a patient with hyper-IgE was found to be similar to that of controls (Wu et al 2015).

The causative mechanism for the immunopathology identified in PGM3-CDG patients may be attributable to altered leukocyte activation, with increased immunoglobulin expression, which boosts allergic and autoimmune responses. Although some defects in glycosylation have been described as increasing immune cell activation (Videira et al 2008), the underlying mechanism in PGM3-CDG patients is still unknown.

As to treatment, the administration of GlcNAc would be a logical approach. Indeed, GlcNAc supplementation restored intracellular UDP-GlcNAc levels in PGM3-deficient cells (Zhang et al 2014), but its therapeutic value in patients remains unknown.

CDG with minor involvement of the immune system

PMM2-CDG

Phosphomannomutase 2 (EC:5.4.2.8) is a cytoplasmic enzyme that converts mannose-6-phosphate into mannose-1-phosphate, the second step of the N-glycosylation pathway. Mutations in phosphomannomutase 2 lead to PMM2-CDG (MIM:212065), the most frequently reported protein N-glycosylation defect (more than 700 patients worldwide). It is a multisystem disorder that affects nearly all organs and systems (Grunewald 2009; Monin et al 2014; Serrano et al 2015). Patients show pronounced psychomotor retardation and a minority of infants and children suffer from recurrent and severe infections that may be lethal (Pérez-Dueñas et al 2009; van de Kamp et al 2007). In approximately 50 % of the patients, typical infections and fever trigger stroke-like episodes (Sparks and Krasnewich 2005). Strømme et al (1991) reported two sibling boys who died from pneumonia at 4 months and 6 years. In the first one, Pneumocystis carinii, a fungus frequently causing pneumonia in immunocompromised hosts, was identified postmortem. The other sibling showed mild hypogammaglobulinemia (IgG and IgA), which can explain the existence of recurrent infections. Imtiaz et al (2000) found among 18 patients, 2 (13 and 12 years) with recurrent infections; in one there was progression to pneumonia ‘on occasions’. In a series of 25 patients with the R141H/F119L genotype (Kjaergaard et al 2001), seven patients died (aged 10 days to 15 years), including four due to infection, either from pneumonia, septic shock or unclassified infection. Blank et al (2006) reported two sibling boys with recurrent episodes of major (Pseudomonas, RSV and Influenza pneumonia, E. coli, S. viridans) infections leading to sepsis as well as minor infections leading to otitis media and chronic mucocutaneous candidiasis. The two siblings received the usual pediatric vaccines at the appropriate times but failed to respond to several of these, and lost protective antibody titers shortly after vaccination. In spite of influenza vaccination, one sibling developed influenza. In both siblings, the varicella vaccine elicited illness and vesicle formation at the vaccination site. Immunological investigation showed normal immunoglobulin levels in the proband and mild decreases of IgG and IgA in the sibling. Neutrophil chemotaxis, which is a key mechanism to fight infections, was reduced by half in the proband. However, the causative mechanism is unclear since the surface expression of adhesion molecules CD11b, CD18, L-selectin, P-selectin glycoprotein ligand-1, platelet endothelial adhesion molecule and sLex on neutrophils was normal, as were physiological rolling studies, lymphocyte proliferation tests and CD3, CD4 and CD8 T cell counts. Both siblings were anergic to skin testing with Candida and mumps. Considering treatment, both received frequent intravenous administration of antibiotics and immunoglobulins, which had some preventive effect. Ong et al (2009) and Verstegen et al (2012) shortly reported a patient with lethal meningitis (caused by Streptococcus pyogenes) and a patient with recurrent bacterial infections, respectively. Interestingly, recurrent/severe infections are not a feature of adults with PMM2-CDG (Monin et al 2014; Stibler et al 1994).

Immunological parameters have been studied in patients with and without recurrent/severe infections. In 15 patients, no correlation was found between recurrent infections and decrease of any serum immunoglobulins (Björklund et al 1997). Moreover, no evidence for hypoglycosylation of serum IgG, which could alter binding to Fc receptors and affect its half-life, was found by Butler et al (2003) in one patient and by Dupré et al (2000) in eight patients. Other proteins related to the immune response have been investigated such as antigen receptors, cytokines, complement factors, complement regulatory proteins and adhesion molecules. A normal expression of CD3, CD19 and CD16 (on lymphocytes), CD33 (on monocytes) and CD54 (on neutrophils) was found. De la Morena-Barrio and colleagues found normal expression of GPI-anchored proteins CD48 (on lymphocytes), CD55 (on monocytes and neutrophils) and CD24 and CD66 (on neutrophils) (de la Morena-Barrio et al 2013), which regulate leukocyte adhesion, polarization and motility.

An increase in the serum levels of cytokines IL-6, IL-8 and IL-10 and decreased levels of IFN-γ was found in three patients with episodes of ascites and pericardial effusion (Truin et al 2008). All cytokines were also present at very high levels in the pericardial and ascites fluids, with the exception of IL-8, which was decreased in ascites fluid. It was proposed that these phenomena might be due to an overload of the ER with underglycosylated proteins. This overload would trigger an over-expression of NF-kB, a protein complex involved in cellular responses to danger associated molecular patterns, initiating an inflammatory response (Heyne et al 1998). Corroborating this notion of altered inflammatory response in PMM-CDG patients, some patients show recurrent fever e causa ignota (de Lonlay et al 2001). Furthermore, an unusual, lethal presentation with congenital thrombocytopenia and hyperferritinemia in the absence of infection was reported, with unexplained macrophage activation revealed in the bone marrow cytology (Noelle et al 2005).

A 2-DE-proteomic analysis of two Spanish PMM2-CDG patients showed abnormalities in serum proteins related to the immune response such as chain B of complement factor C3c, α2-macroglobulin, haptoglobin and fibrinogen (Richard et al 2009). Surface-expressed sialoglycans of EBV-transformed cell lines derived from patient’s B cells expressed less α2,6 sialylated glycans and bound less CD22 lectin (Bergmann et al 1998), specific for α2,6 sialylated lactosamines and known to modulate B cell receptor mediated signaling. This could not be explained by enzyme assays showing a normal intracellular α2,6 sialyltransferase activity. Because of its role in innate immune response, intercellular adhesion molecule-1 (ICAM-1) was studied in fibroblasts from PMM2-CDG patients, and found decreased (He et al 2014).

Van Dijk et al (2001) found an increased α3-fucosylation (sLex determinant) and an increased branching of the acute-phase proteins α1-acid glycoprotein, α1-anti-chymotrypsin and α1-protease inhibitor. There was also a tendency to an increased α3-fucosyltransferase activity in the plasma. An increased expression of sLex determinant on α1-acid glycoprotein has been shown to indicate a hepatic inflammatory reaction (De Graaf et al 1993), which is of the chronic type when there is an increased branching of the glycans (Van Dijk et al 1998).

Overall, some PMM2-CDG patients show recurrent and lethal infections. This might be related to a number of events such as decreased immunoglobulin levels, leukocyte migration and immune-related serum proteins.

MAN1B1-CDG

Mannosyl-oligosaccharide 1,2-α-mannosidase (EC:3.2.1.113) trims a single α1,2-linked mannose residue from Man9GlcNAc2 in the Golgi. Mutations in MAN1B1, causing MAN1B1-CDG, have first been reported in 2013. The main features in 31 patients are developmental/intellectual disability, hypotonia, behavioural problems, facial dysmorphism and truncal obesity (Rymen et al 2013; Van Scherpenzeel et al 2014). These patients do not show an increased incidence of inflammations or infections. Serum immunoglobulins are quantitatively normal but show glycan abnormalities: high mannosylated hybrid glycans, mostly penta- and hexamannosylglycans with one or two antennas, and one or two galactose residues. There is also an increase of sLex-containing glycans, that are also found on acute phase proteins in inflammation and cancer (Saldova et al 2015), but there is no evidence for the latter conditions in MAN1B1-CDG. Patients’ fibroblasts exhibit a marked Golgi aberrant morphology (Rymen et al 2013) and the reason for the increase of the sLex epitope may be the disruption of the Golgi morphology in these patients (Saldova et al 2015). This may lead to dysregulation of the Golgi pH and secondary defective glycosylation. Glycosylation of immunoglobulin G is important for its function (Saldova et al 2015) but the precise impact of the above mentioned glycosylation changes on IgG function is not known.

COG6-CDG

The conserved oligomeric Golgi complex (COG) is composed of eight subunits, named COG1 to COG8. This complex is thought to be involved in the maintenance of the Golgi glycosylation machinery (Pokrovskaya et al 2011). Mutations have been described in each subunit except subunit 3.

COG6-CDG (MIM:606977) has been reported in 17 patients with the following core features: liver involvement, microcephaly, developmental disability, recurrent infections, early lethality, hypohidrosis predisposing to hyperthermia and hyperkeratosis (Huybrechts et al 2012; Lübbehusen et al 2010; Rymen et al 2015; Shaheen et al 2013). Recurrent infections were reported in 8/17 patients. Among these, one patient showed combined immunodeficiency, another B and T cell defects and neutrophil dysfunction, and still another patient marked monocytosis and a deficient polysaccharide antibody response.

ALG1-CDG

GDP-Man:GlcNAc2-PP-Dol mannosyltransferase (mannosyltransferase 1; EC:2.4.1.142) catalyzes the attachment of the first of five mannoses in the N-glycan formed at the cytosolic side of the ER.

Mutations in the gene coding for this enzyme cause ALG1-CDG (MIM:608540). The 19 published patients showed predominantly dysmorphism and neurological involvement. Eight of them suffered from severe infections and/or frequent episodes of unexplained fever (Grubenmann et al 2004; Morava et al 2012). In two of these patients, serum IgG was severely decreased, most probably secondary to nephrotic syndrome and protein-losing enteropathy, respectively (Kranz et al 2004; Rohlfing et al 2014).

MGAT2-CDG

UDP-N-acetylglucosamine:α-6-D-mannoside β-1,2-N-acetylglucosaminyltransferase (N-acetylglucosaminyltransferase II, EC:2.4.1.143) catalyzes the attachment of the first of two GlcNAc to the trimannosylcore in the Golgi. Only 12 patients (including a family with nine subjects) have been reported with MGAT2-CDG (MIM:212066). Their phenotype was characterized by severe neurological, skeletal and intestinal involvement (de Cock and Jaeken 2009; Jaeken 2012; Jaeken et al 1993; Ramaekers et al 1991). One patient showed recurrent respiratory infections (Jaeken et al 1994) and died at 18 years of age with respiratory insufficiency and hypercapnia. He showed transiently decreased levels of serum IgG (up to the age of 6 years), decreased total hemolytic complement (CH50) assay, C2, C3a, and increased C3d. Additional investigations showed IgG with absent light chain glycosylation, and abnormal heavy chain glycosylation (Butler et al 2003). As mentioned, altered IgG glycosylation compromises the stability, half-life and its receptor-mediated functions. Complement factor H-related protein was found to be missing as well as an isoform of the complement C4 γ chain. Thus, the infections observed in some of the MGAT2-CDG patients are associated with defects in IgG and complement protein levels.

DOLK-CDG

Dolichol kinase (EC:2.7.1.108) catalyzes the last step in the synthesis of dolichol phosphate. The latter is an essential glycosyl carrier lipid for O- and C-mannosylation, for protein N-glycosylation and for the biosynthesis of GPI anchors in the ER. DOLK-CDG (MIM:610768) has been reported in at least 18 patients (Lieu et al 2013). Patients presented various combinations and degrees of cardiac, neurological and skin involvement, including dysmorphism. The first report was on four patients. They died in infancy, one at 6 months of age from a severe pulmonary infection, caused by the respiratory syncytial virus (Kranz et al 2007a, b). Kapusta et al (2013) reported on cardiac pathology (from mild dilatation to overt heart failure) in nine patients from three families. One patient showed cardiac failure during infections. Another patient improved after heart transplantation, except for recurrent herpetic infections, but died unexpectedly 1.5 year after transplantation. Still another patient suffered from frequent infections with severe neutropenia and microcytic anemia. Although there is little evidence supporting immunopathology, some patients suffered from recurrent and severe infections and most of them had leukopenia without thrombocytopenia (Kapusta et al 2013), suggesting an impairment in leukocyte production or differentiation.

PIGY-CDG

PIG-Y is the smallest subunit of the GPI-N-acetylglucosaminyltransferase (GPI-GnT) (EC:2.4.1.198), that catalyzes the first step in the biosynthesis of the GPI anchors. The four reported children with PIGY mutations presented a severe to moderately severe phenotype. One had necrotizing enterocolitis and chronic lung disease and died at 2 years of age from a respiratory infection (Ilkovski et al 2015). No immunological parameters have been reported.

GALNT3-CDG

The GALNT3 gene encodes GalNAc‐T3 (EC:2.4.1.41), one of the several UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferases, responsible for the first O-glycosylation step, i.e. the transfer of GalNAc to a serine or threonine residue. Mutations in GALNT3 are associated with familial tumoral calcinosis (FTC), characterized by the development of multiple calcified masses in periarticular soft tissues. Immune disorders, including vasculitis and arthritis, have been associated with FTC (Metzker et al 1988). GALNT3 gene mutation has been also associated with chronic recurrent multifocal osteomyelitis (Demellawy et al 2015), which is regarded as an auto-inflammatory disease without infection. Further work is needed to explore the relationship between auto-immune/inflammatory disease and GALNT3 mutations.

Secondary CDG with immunological involvement: the case of galactosemia

Galactosemia (MIM:230400) is caused by mutations in the GALT gene, coding for galactose 1-phosphate uridyltransferase (EC:2.7.7.12). It is a primary defect in galactose metabolism that causes a secondary glycosylation disorder by a yet incompletely understood mechanism. Patients may appear normal at birth, but within a few days of starting breast or formula milk feeding they usually develop a life-threatening illness with hepatic, renal and cerebral involvement. Treatment consists in the exclusion of galactose and thus also of lactose from the diet (Bosch 2006).

There is an increased incidence of sepsis, which is one of the most important causes of early death. Sepsis is mostly due to E. coli but also to other bacteria (Berry 1993, 2000; Waggoner et al 1990), as well as to fungi (Verma et al 2010). The cause of this increased sepsis incidence is not clear but Kobayashi et al (1983) have reported a depressed function of galactosemia patient neutrophils when incubated with galactose, which was greater in neonatal neutrophils than in adult neutrophils. According to another study, the bactericidal activity, including NADPH activity in blood lymphocytes of galactosemia patients is impaired (Al-Essa et al 2013; Litchfield and Wells 1978). Liver impairment and cirrhosis, a typical complication in untreated patients, may also boost E. coli sepsis (Gustot et al 2009). It has also been demonstrated that patients IgG show abnormal glycosylation (Coss et al 2014), but it is uncertain whether this plays a role in the predisposition to infection (Maratha et al 2016). An unusual presentation of galactosemia is hemophagocytic lymphohistiocytosis. This is a clinical syndrome resulting from uncontrolled proliferation of activated lymphocytes and histiocytes that secrete large quantities of inflammatory cytokines (Kundak et al 2012).

Hyperglycosylation and immunological involvement

The present work focuses on hypoglycosylation defects, mostly due to loss-of-glycosylation events, but it seems logical that disruption of immune regulation also occurs when gain-of-glycosylation mutations occur. An example of this is interferon receptor hyperglycosylation, due to a mutation introducing a new asparagine residue (T168N IFNGR2), that leads to a novel N-glycosylation site. This mutation renders the receptor unable to respond to IFN-γ engagement, predisposing to intracellular bacterial infections (Vogt et al 2005). It is most probable that other, yet unknown, glycosylation sites are introduced by mutations in genes coding for other key immunological proteins.

Conclusions

Nearly 10 % of all described CDG show some degree of immunological involvement. These defective proteins are distributed evenly within the cell (Fig. 1). Since many immunological factors/compounds are glycosylated, it is somewhat surprising that there is not a greater immunological impact caused by CDG. A reason for this may be the existence of compensatory immune mechanisms. Another explanation might be that many patients have a partial genetic defect, so that there is only a mild immunological involvement not leading to clinical expression. The clinical immunological spectrum comprises severe/recurrent and local/generalized infections (bacterial, viral, fungal), autoimmunity, allergy and atopy. A basic treatment for the ‘immunological CDG’ is only available for SLC35C1-CDG, and only for patients with particular mutations. In the group of CDG with ‘minor’ immunological involvement, it is not always clear whether infections are due to an immunological or to another problem. There are many factors contributing to the ultimate phenotype, ranging from different mutations to the personal background. While there are no statistics on the prevalence of bacterial infections in CDG patients, it becomes evident that glycosylation defects may have a major impact on immune homeostasis and defense against pathogens. Bacterial colonization is assumed to be facilitated by impaired glycosylation on host cells. A lack of functional complement proteins or dampened innate immune responses may explain the increased susceptibility to pathogens. It is also relevant to recall that the recognition of immunoglobulins by Fc receptors is dependent on a proper glycosylation of the immunoglobulins and its receptors.

Interestingly, CDG where increased pro-inflammatory cytokines were identified such as PMM2-CDG, also manifest frequent stroke-like episodes. Indeed, recent findings support the assumption of a pro-inflammatory process, with an altered pattern of circulating pro-inflammatory cytokines in pediatric stroke episodes (Buerki et al 2016). To obtain a better insight into this matter, it would be interesting to perform a complete immunological check-up in all CDG patients with or without a clinical immunological problem. The association of these data with characteristic features of CDG may reveal relevant pathological mechanisms. Further studies are necessary to understand the molecular pathways affected in CDG patients and, particularly, the immunological mechanisms compromised by the absence of proper glycosylation.