Association between Neu5Gc carbohydrate and serum antibodies against it provides the molecular link to cancer: French NutriNet-Santé study.

BACKGROUND: High consumption of red and processed meat is commonly associated with increased cancer risk, particularly colorectal cancer. Antibodies against the red meat-derived carbohydrate N-glycolylneuraminic acid (Neu5Gc) exacerbate cancer in "human-like" mice. Human anti-Neu5Gc IgG and red meat are both independently proposed to increase cancer risk, yet how diet affects these antibodies is largely unknown. METHODS: We used world global data to demonstrate that colorectal cancer incidence and mortality are associated with increased national meat consumption. In a well-defined large cohort, we used glycomics to measure daily Neu5Gc intake from red meat and dairy, and investigated serum as well as affinity-purified anti-Neu5Gc antibodies. Based on 24-h dietary records, daily Neu5Gc intake was calculated for 19,621 subjects aged ≥ 18 years of the NutriNet-Santé study. Serum and affinity-purified anti-Neu5Gc antibodies were evaluated by ELISA and glycan microarrays in representative 120 individuals, each with at least eighteen 24-h dietary records (aged 45-60, Q1-Q4; aged > 60, Q1 and Q4; 10 men/women per quartile). RESULTS: We found that high-Neu5Gc diet, gender, and age affect the specificity, levels, and repertoires of anti-Neu5Gc IgG immune responses, but not their affinity. Men consumed more Neu5Gc than women, mostly from red meat (p = 0.0015), and exhibited higher overall serum anti-Neu5Gc IgG levels by ELISA (3.94 ng/µl versus 2.22 ng/µl, respectively; p = 0.039). Detailed glycan microarray analysis against 56 different glycans revealed high Neu5Gc-specificity with increased anti-Neu5Gc IgG and altered repertoires, associated with higher consumption of Neu5Gc from red meat and cow dairy. Affinity purification of serum anti-Neu5Gc antibodies revealed increased levels and biased array repertoire patterns, without an increase in antibody affinity, in individuals consuming higher Neu5Gc levels. Furthermore, in a high-meat diet, antibody diversity patterns on glycan microarrays shifted towards Neu5Gcα3-linked glycans, increasing the α3/α6-glycans ratio score. CONCLUSIONS: We found a clear link between the levels and repertoire of serum anti-Neu5Gc IgG and Neu5Gc intake from red meat and dairy. These precise rational methodologies allowed to develop a Gcemic index to simplify the assessment of Neu5Gc in foods that could potentially be adapted for dietary recommendations to reduce cancer risk.

Microarray Analysis of Oligosaccharide-Mediated Multivalent Carbohydrate-Protein Interactions and Their Heterogeneity.

Carbohydrate-protein interactions (CPIs) are involved in a wide range of biological phenomena. Hence, the characterization and presentation of carbohydrate epitopes that closely mimic the natural environment is one of the long-term goals of glycosciences. Inspired by the multivalency, heterogeneity and nature of carbohydrate ligand-mediated interactions, we constructed a combinatorial library of mannose and galactose homo- and hetero-glycodendrons to study CPIs. Microarray analysis of these glycodendrons with a wide range of biologically important plant and animal lectins revealed that oligosaccharide structures and heterogeneity interact with each other to alter binding preferences.

The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration.

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

Synthetic heparan sulfate ligands for vascular endothelial growth factor to modulate angiogenesis.

We report the discovery of a potential heparan sulfate (HS) ligand to target several growth factors using 13 unique HS tetrasaccharide ligands. By employing an HS microarray and SPR, we deciphered the crucial structure-binding relationship of these glycans with the growth factors BMP2, VEGF165, HB-EGF, and FGF2. Notably, GlcNHAc(6-O-SO3-)-IdoA(2-O-SO3-) (HT-2,6S-NAc) tetrasaccharide showed strong binding with the VEGF165 growth factor. In vitro vascular endothelial cell proliferation, migration and angiogenesis was inhibited in the presence of VEGF165 and HT-2,6S-NAc or HT-6S-NAc, revealing the potential therapeutic role of these synthetic HS ligands.

Heparan Sulfate Mimetics Differentially Affect Homologous Chemokines and Attenuate Cancer Development.

Achieving selective inhibition of chemokine activity by structurally well-defined heparan sulfate (HS) or HS mimetic molecules can provide important insights into their roles in individual physiological and pathological cellular processes. Here, we report a novel tailor-made HS mimetic, which furnishes an exclusive iduronic acid (IdoA) scaffold with different sulfation patterns and oligosaccharide chain lengths as potential ligands to target chemokines. Notably, highly sulfated-IdoA tetrasaccharide (I-45) exhibited strong binding to CCL2 chemokine thereby blocking CCL2/CCR2-mediated in vitro cancer cell invasion and metastasis. Taken together, IdoA-based HS mimetics offer an alternative HS substrate to generate selective and efficient inhibitors for chemokines and pave the way to a wide range of new therapeutic applications in cancer biology and immunology.

Glycosylated Biotherapeutics: Immunological Effects of N-Glycolylneuraminic Acid.

The emerging field of biotherapeutics provides successful treatments for various diseases, yet immunogenicity and limited efficacy remain major concerns for many products. Glycosylation is a key factor determining the pharmacological properties of biotherapeutics, including their stability, solubility, bioavailability, pharmacokinetics, and immunogenicity. Hence, an increased attention is directed at optimizing the glycosylation properties of biotherapeutics. Currently, most biotherapeutics are produced in non-human mammalian cells in light of their ability to produce human-like glycosylation. However, most mammals produce the sialic acid N-glycolylneuraminic acid (Neu5Gc), while humans cannot due to a specific genetic defect. Humans consume Neu5Gc in their diet from mammalian derived foods (red meat and dairy) and produce polyclonal antibodies against diverse Neu5Gc-glycans. Moreover, Neu5Gc can metabolically incorporate into human cells and become presented on surface or secreted glycans, glycoproteins, and glycolipids. Several studies in mice suggested that the combination of Neu5Gc-containing epitopes and anti-Neu5Gc antibodies could contribute to exacerbation of chronic inflammation-mediated diseases (e.g., cancer, cardiovascular diseases, and autoimmunity). This could potentially become complicated with exposure to Neu5Gc-containing biotherapeutics, bio-devices or xenografts. Indeed, Neu5Gc can be found on various approved and marketed biotherapeutics. Here, we provide a perspective review on the possible consequences of Neu5Gc glycosylation of therapeutic protein drugs due to the limited published evidence of Neu5Gc glycosylation on marketed biotherapeutics and studies on their putative effects on immunogenicity, drug efficacy, and safety.

Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer.

Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19-9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.

ABO Antigens Active Tri- and Disaccharides Microarray to Evaluate C-type Lectin Receptor Binding Preferences.

Understanding blood group antigen binding preferences for C-type lectin receptors holds promise for modulating immune responses, since several Gram-negative bacteria express blood group antigens as molecular mimicry to evade immune responses. Herein, we report the synthesis of ABO blood group antigen active tri and disaccharides to investigate the binding specificity with various C-type lectin receptors using glycan microarray. The results of binding preferences show that distinct glycosylation on the galactose and fucose motifs are key for C-type lectin receptor binding and that these interactions occur in a Ca2+-dependent fashion.