LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Functional genomics identifies N-acetyllactosamine extension of complex N-glycans as a mechanism to evade lysis by natural killer cells.

Natural killer (NK) cells are primary defenders against cancer precursors, but cancer cells can persist by evading immune surveillance. To investigate the genetic mechanisms underlying this evasion, we perform a genome-wide CRISPR screen using B lymphoblastoid cells. SPPL3, a peptidase that cleaves glycosyltransferases in the Golgi, emerges as a top hit facilitating evasion from NK cytotoxicity. SPPL3-deleted cells accumulate glycosyltransferases and complex N-glycans, disrupting not only binding of ligands to NK receptors but also binding of rituximab, a CD20 antibody approved for treating B cell cancers. Notably, inhibiting N-glycan maturation restores receptor binding and sensitivity to NK cells. A secondary CRISPR screen in SPPL3-deficient cells identifies B3GNT2, a transferase-mediating poly-LacNAc extension, as crucial for resistance. Mass spectrometry confirms enrichment of N-glycans bearing poly-LacNAc upon SPPL3 loss. Collectively, our study shows the essential role of SPPL3 and poly-LacNAc in cancer immune evasion, suggesting a promising target for cancer treatment.

Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galß1-3GlcNAcß1-3Galß1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galß1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection.

Recent human H3N2 influenza A viruses have evolved to employ elongated glycans terminating in α2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by (humanized) Madin-Darby Canine Kidney cells, which are commonly employed to propagate influenza A virus, resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAcs), would result in improved A/H3N2 propagation. Stable overexpression of ß-1,3-N-acetylglucosaminyltransferase and ß-1,4-galactosyltransferase 1 in Madin-Darby Canine Kidney and "humanized" Madin-Darby Canine Kidney cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the ß-1,3-N-acetylglucosaminyltransferase and/or ß-1,4-galactosyltransferase 1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on "humanized" Madin-Darby Canine Kidney-ß-1,3-N-acetylglucosaminyltransferase cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 influenza A viruses require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

Trimming Crystallizable Fragment (Fc) Glycans Enables the Direct Enzymatic Transfer of Biomacromolecules to Antibodies as Therapeutics.

Glycoengineering has provided powerful tools to construct site-specific antibody conjugates. However, only small-molecule payloads can be directly transferred to native or engineered antibodies using existing glycoengineering strategies. Herein, we demonstrate that reducing the complexity of crystallizable fragment (Fc) glycans could dramatically boost the chemoenzymatic modification of immunoglobulin G (IgG) via an engineered fucosyltransferase. In this platform, antibodies with Fc glycans engineered to a simple N-acetyllactosamine (LacNAc) disaccharide are successfully conjugated to biomacromolecules, such as oligonucleotides and nanobodies, in a single step within hours. Accordingly, we synthesized an antibody-conjugate-based anti-human epidermal growth factor receptor 2 (HER2)/ cluster of differentiation 3 (CD3) bispecific antibody and used it to selectively destroy patient-derived cancer organoids by reactivating endogenous T lymphocyte cells (T cells) inside the organoid. Our results highlight that this platform is a general approach to construct antibody-biomacromolecule conjugates with translational values.

LacNAc modification in bone marrow stromal cells enhances resistance of myelodysplastic syndrome cells to chemotherapeutic drugs.

Chemotherapeutic drugs are used routinely for treatment for myelodysplastic syndrome (MDS) patients but are ineffective in a substantial proportion of patients. Abnormal hematopoietic microenvironments, in addition to spontaneous characteristics of malignant clones, contribute to ineffective hematopoiesis. In our study, we found expression of enzyme ß1,4-galactosyltransferase 1 (ß4GalT1), which regulates N-acetyllactosamine (LacNAc) modification of proteins, is elevated in bone marrow stromal cells (BMSCs) of MDS patients, and also contributes to drug ineffectiveness through a protective effect on malignant cells. Our investigation of the underlying molecular mechanism revealed that ß4GalT1-overexpressing BMSCs promoted MDS clone cells resistant to chemotherapeutic drugs and also showed enhanced secretion of cytokine CXCL1 through degradation of tumor protein p53. Chemotherapeutic drug tolerance of myeloid cells was inhibited by application of exogenous LacNAc disaccharide and blocking of CXCL1. Our findings clarify the functional role of ß4GalT1-catalyzed LacNAc modification in BMSCs of MDS. Clinical alteration of this process is a potential new strategy that may substantially enhance effectiveness of therapies for MDS and other malignancies, by targeting a niche interaction.

Immune complex-induced haptokinesis in human non-classical monocytes.

Formation and deposition of immune complexes (ICs) are hallmarks of various autoimmune diseases. Detection of ICs by IC receptors on leukocytes induces downstream signaling and shapes the local immune response. In many cases the pathological relevance of ICs is not well understood. We here show that ICs induce a distinct migratory response, i.e. haptokinesis in 6-sulfo LacNAc+ monocytes (slanMo) and in non-classical monocytes (ncMo) but not in intermediate (imMo) and classical monocytes (cMo). Using live imaging combined with automated cell tracking, we show that the main features of IC-dependent haptokinesis are elongation of the cell body, actin polarization at the leading edge, and highly directional migration. We find that CD16-dependent signaling mediates haptokinesis as blocking of CD16 or blocking SYK-signaling inhibited the migratory response. The activity of the metalloproteinase ADAM17 also modifies IC-dependent haptokinesis, likely at least partially via cleavage of CD16. Furthermore, using matrices with defined ligand spacing, we show that ligand density impacts the magnitude of the migratory response. Taken together, we have demonstrated that ICs induce a specific migratory response in ncMo but not in other monocyte subsets. Therefore, our work lays the groundwork for the investigation of IC-dependent haptokinesis in ncMo as a potential pathomechanism in IC-mediated autoimmune diseases.

Biochemical characterization and cytotoxicity of polylactosamine-extended N-glycans binding isolectins from the mushroom Hericium erinaceus.

The mushroom Hericium erinaceus expresses isolectins with different glycan binding specificities; of these, the ricin B-like lectin HEL1 and HEL2 (HEL2a and HEL2b) can bind fucosylated N-glycans and core 1 O-glycans, respectively. However, other lectin-like protein-coding transcripts detected in the H. erinaceus transcriptome, named HEL3, remain to be characterized. Therefore, in this study, the expression levels of all these isolectins genes were compared to characterize the molecular and biochemical properties of these carbohydrate-binding proteins. Low expression genes encoding putative cytolysin proteins, HEL3a and HEL3b, were identified. Bioinformatics analyses revealed that these proteins shared highly homologous structures and carbohydrate-binding residues with other mushroom lectins. Further, their recombinant proteins, rHEL3a and rHEL3b showed an octamer composed of identical 17 kDa subunits under non-denaturing conditions and a slightly basic isoelectric point value of approximately 8.3. The hemagglutination activity of these isolectins was strongly inhibited by glycoproteins rather than free glycans. Interestingly, glycan-binding profiles showed that rHEL3 isolectins interacted with most polylactosamine (poly-LacNAc)-extended N-glycans with relatively low binding activity. Isothermal titration calorimetry also revealed that these recombinant lectins have different binding capacities toward N-glycan-containing glycoproteins. Further, treatment with different concentrations of rHEL3 lectins showed cytotoxic effects in K562, UACC62, and CHO model cell lines, which express poly-LacNAc glycans, confirmed by inhibition of proliferation. Overall, these biochemical properties indicate that rHEL3 isolectins may be used as unique lectins for detecting poly-LacNAc-extended glycans, which are known to be over-expressed in leukemia or metastatic melanoma cells, in cancer diagnostic assays and anti-cancer therapies.

The synergistic function of long and short forms of ß4GalT1 in p53-mediated drug resistance in bladder cancer cells.

ß1,4-galactosyltransferase-1 (ß4GalT1) is a type II membrane protein that catalyzes the transfer of galactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc) and forms a LacNAc structure. ß4GalT1 has a long form (termed ß4GalT1-L) and a short form (termed ß4GalT1-S) in mammalian cells. Although ß4GalT1 has been proven to play an important role in many biological and pathological processes, such as differentiation, immune responses and cancer development, the different functions of the two ß4GalT1 forms remain ambiguous. In this study, we demonstrated that total ß4GalT1 was upregulated in bladder cancer. Overexpression of ß4GalT1-S, but not ß4GalT1-L, increased drug resistance in bladder epithelial cells by upregulating p53 expression. Glycoproteomic analysis revealed that the substrate specificities of the two ß4GalT1 forms were different. Among the LacNAcylated proteins, the E3 ligase MDM2 could be preferentially modified by ß4GalT1-L compared to ß4GalT1-S, and this modification could increase the binding of MDM2 and p53 and further facilitate the degradation of p53. Our data proved that the two forms of ß4GalT1 could synergistically regulate p53-mediated cell survival under chemotherapy treatment. These results provide insights into the role of ß4GalT1-L and ß4GalT1-S and suggest their differentially important implications in the development of bladder cancer.

From structure to function - Ligand recognition by myeloid C-type lectin receptors.

The relevance of protein-glycan interactions in immunity has long been underestimated. Yet, the immune system possesses numerous classes of glycan-binding proteins, so-called lectins. Of specific interest is the group of myeloid C-type lectin receptors (CLRs) as they are mainly expressed by myeloid cells and play an important role in the initiation of an immune response. Myeloid CLRs represent a major group amongst pattern recognition receptors (PRRs), placing them at the center of the rapidly growing field of glycoimmunology. CLRs have evolved to encompass a wide range of structures and functions and to recognize a large number of glycans and many other ligands from different classes of biopolymers. This review aims at providing the reader with an overview of myeloid CLRs and selected ligands, while highlighting recent insights into CLR-ligand interactions. Subsequently, methodological approaches in CLR-ligand research will be presented. Finally, this review will discuss how CLR-ligand interactions culminate in immunological functions, how glycan mimicry favors immune escape by pathogens, and in which way immune responses can be affected by CLR-ligand interactions in the long term.

A Redox-Controlled Substrate Engineering Strategy for Site-Specific Enzymatic Fucosylation.

Fucosylation is one of the most common modifications of oligo-N-acetyllactosamine (oligo-LacNAc) glycans. However, none of known fucosyltransferases (FucTs) could install the α1,3-linked fucose to the oligo-LacNAc substrates in a site-specific manner. Here, we report a facile and general redox-controlled substrate engineering strategy for the site-specific α1,3-fucosylation of complex glycans containing multiple LacNAc units. This strategy takes advantage of an operationally simple oxidation enzyme module by using galactose oxidase (GOase) to convert the LacNAc unit into oxidized C6'-aldehyde LacNAc sequence, which is not a good substrate for recombinant α1,3-FucT from Helicobacter pylori strain 26695 (Hpα1,3FucT), enabling the site-specific α1,3-fucosylation at intact LacNAc sites. The general applicability and robustness of this strategy were demonstrated by the synthesis of a variety of structurally well-defined fucosides of linear and branched O- and N-linked glycans.

One-step synthesis of site-specific antibody-drug conjugates by reprograming IgG glycoengineering with LacNAc-based substrates.

Glycosite-specific antibody‒drug conjugatess (gsADCs), harnessing Asn297 N-glycan of IgG Fc as the conjugation site for drug payloads, usually require multi-step glycoengineering with two or more enzymes, which limits the substrate diversification and complicates the preparation process. Herein, we report a series of novel disaccharide-based substrates, which reprogram the IgG glycoengineering to one-step synthesis of gsADCs, catalyzed by an endo-N-acetylglucosaminidase (ENGase) of Endo-S2. IgG glycoengineering via ENGases usually has two steps: deglycosylation by wild-type (WT) ENGases and transglycosylation by mutated ENGases. But in the current method, we have found that disaccharide LacNAc oxazoline can be efficiently assembled onto IgG by WT Endo-S2 without hydrolysis of the product, which enables the one-step glycoengineering directly from native antibodies. Further studies on substrate specificity revealed that this approach has excellent tolerance on various modification of 6-Gal motif of LacNAc. Within 1 h, one-step synthesis of gsADC was achieved using the LacNAc-toxin substrates including structures free of bioorthogonal groups. These gsADCs demonstrated good homogeneity, buffer stability, in vitro and in vivo anti-tumor activity. This work presents a novel strategy using LacNAc-based substrates to reprogram the multi-step IgG glycoengineering to a one-step manner for highly efficient synthesis of gsADCs.

6-Sulfo LacNAc monocytes are quantitatively and functionally disturbed in systemic sclerosis patients.

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis, microangiopathy, and autoantibodies. We previously reported that circulating follicular helper T (cTfh) cells are increased in SSc and induce plasmablast differentiation. However, mechanisms leading to cTfh cell expansion and activation in SSc remain to be established. Tfh cells require IL-12 for their expansion and differentiation. 6-Sulfo LacNAc monocytes (slanMo), a subset of monocytes, have a higher capacity to produce IL-12 and to induce CD4+ T cell proliferation in comparison with dendritic cells (DC) or classical monocytes. The aim of this study was to perform a quantitative and functional analysis of monocytes and DC and to correlate them with cTfh cell expansion and clinical manifestations in SSc. Using flow cytometry, we analyzed different monocyte subsets including slanMo and DC from 36 SSc patients and 26 healthy controls (HC). In vitro culture experiments of sorted slanMo were performed for functional analysis and cytokine production. We observed that slanMo, intermediate and non-classical monocytes were increased in SSc in comparison with HC. Furthermore, the increase in slanMo cells was more potent in patients with diffuse SSc. We observed a significant positive correlation between slanMo and cTfh cell levels in SSc patients but not in HC. Other monocyte subsets did not correlate with cTfh cell expansion. In addition, we observed that in vitro, slanMo cells from SSc patients produced less IL-12 than slanMo from HC. SlanMo are increased in SSc and may participate in the activation of cTfh cells in SSc.

Method for Identifying Galectin Ligands on Lymphocyte Membrane Glycoproteins.

Glycosylation is one of the most common protein posttranslational modifications. Most human lymphocyte membrane receptors are modified by diverse glycan structures, and functional studies have indicated that a family of glycan-binding proteins, galectins, can significantly modulate lymphocyte development and function by interacting with these glycans. Several galectins have a varying degree of affinity for the N-acetyllactosamine (LacNAc) disaccharide, and some critical lymphocyte receptors can be modified by glycan structures carrying this motif. However, the site-specific glycan composition on primary lymphocyte membrane receptors in healthy individuals is largely limited. The main reason for the limitation is low abundance of available material from a single donor and compositional heterogeneity in glycan structures that can potentially modify a protein. Donor-dependent variability in N-glycan structures on CD16a isolated from primary NK cells of healthy human donors was recently reported. NK cell CD16a is glycosylated at five N-glycosylation sites, and two of the five sites are modified, almost exclusively, by N-glycans with multiple LacNAc repeats which can serve as ligands for endogenous galectins. Thus, the protocol described in this section can be utilized to identify galectin ligands at specific glycosylation sites of endogenous membrane receptor from circulating primary human lymphocytes.

An Egg-Derived Sulfated N-Acetyllactosamine Glycan Is an Antigenic Decoy of Influenza Virus Vaccines.

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess different glycosylation patterns than viruses circulating among humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived glycan that is an antigenic decoy, with egg-binding MAbs reacting with a sulfated N-acetyllactosamine (LacNAc). Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken-derived cells, suggesting that only egg-grown vaccines can induce antiegg antibodies. Notably, antibodies against the egg antigen utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and had a simple heavy-chain complementarity-determining region 3. By analyzing a public data set of influenza virus vaccine-induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

Safety and Modulatory Effects of Humanized Galacto-Oligosaccharides on the Gut Microbiome.

Complex dietary carbohydrate structures including ß(1-4) galacto-oligosaccharides (GOS) are resistant to digestion in the upper gastrointestinal (GI) tract and arrive intact to the colon where they benefit the host by selectively stimulating microbial growth. Studies have reported the beneficial impact of GOS (alone or in combination with other prebiotics) by serving as metabolic substrates for modulating the assembly of the infant gut microbiome while reducing GI infections. N-Acetyl-D-lactosamine (LacNAc, Galß1,4GlcNAc) is found in breast milk as a free disaccharide. This compound is also found as a component of human milk oligosaccharides (HMOs), which have repeating and variably branched lactose and/or LacNAc units, often attached to sialic acid and fucose monosaccharides. Human glycosyl-hydrolases do not degrade most HMOs, indicating that these structures have evolved as natural prebiotics to drive the proper assembly of the infant healthy gut microbiota. Here, we sought to develop a novel enzymatic method for generating LacNAc-enriched GOS, which we refer to as humanized GOS (hGOS). We showed that the membrane-bound ß-hexosyl transferase (rBHT) from Hamamotoa (Sporobolomyces) singularis was able to generate GOS and hGOS from lactose and N-Acetyl-glucosamine (GlcNAc). The enzyme catalyzed the regio-selective, repeated addition of galactose from lactose to GlcNAc forming the ß-galactosyl linkage at the 4-position of the GlcNAc and at the 1-position of D-galactose generating, in addition to GOS, LacNAc, and Galactosyl-LacNAc trisaccharides which were produced by two sequential transgalactosylations. Humanized GOS is chemically distinct from HMOs, and its effects in vivo have yet to be determined. Thus, we evaluated its safety and demonstrated the prebiotic's ability to modulate the gut microbiome in 6-week-old C57BL/6J mice. Longitudinal analysis of gut microbiome composition of stool samples collected from mice fed a diet containing hGOS for 5 weeks showed a transient reduction in alpha diversity. Differences in microbiome community composition mostly within the Firmicutes phylum were observed between hGOS and GOS, compared to control-fed animals. In sum, our study demonstrated the biological synthesis of hGOS, and signaled its safety and ability to modulate the gut microbiome in vivo, promoting the growth of beneficial microorganisms, including Bifidobacterium and Akkermansia.

Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery.

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called "targeting by timing." Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC-MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of "glycosylation by timing" during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.

Integrated Glycome Strategy for Characterization of Aberrant LacNAc Contained N-Glycans Associated With Gastric Carcinoma.

Aberrant glycosylation is not only a feature of malignant cell transformation, but also plays an important role in metastasis. In the present study, an integrated strategy combining the lectin microarrays and lectin cytochemistry was employed to investigate and verify the altered glycopatterns in gastric cancer (GC) cell lines as well as resected tumor specimens from matched tissue sets of 46 GC patients. Subsequently, lectin-mediated affinity capture glycoproteins, and MALDI-TOF/TOF-MS were employed to further acquire precise structural information of the altered glycans. According to the results, the glycopatterns recognized by 10 (e.g., ACA, MAL-I, and ConA) and 3 lectins (PNA, MAL-I, and VVA) showed significantly variations in GC cells and tissue compared to their corresponding controls, respectively. Notably, the relative abundance of Galß-1,4GlcNAc (LacNAc) recognized by MAL-I exhibited a significant increase in GC cells (p < 0.001) and tissue from patients at stage II and III (p < 0.05), and a significant increase in lymph node positive tumor cases, compared with lymph node negative tumor cases (p < 0.05). More LacNAc contained N-glycans were characterized in tumor sample with advanced stage compared to corresponding control. Moreover, there were 10 neo-LacNAc-contained N-glycans (e.g., m/z 1625.605, 1803.652, and 1914.671) only presented in GC tissue with advanced stage. Among these, six N-glycans were modified with sialic acid or fucose based on LacNAc to form sialylated N-glycans or lewis antigens, respectively. Our results revealed that the aberrant expression of LacNAc is a characteristic of GC, and LacNAc may serve as a scaffold to be further modified with sialic acid or fucose. Our findings provided useful information for us to understand the development of GC.

Expedient assembly of Oligo-LacNAcs by a sugar nucleotide regeneration system: Finding the role of tandem LacNAc and sialic acid position towards siglec binding.

Sialosides containing (oligo-)N-acetyllactosamine (LacNAc, Galß(1,4)GlcNAc) as core structure are known to serve as ligands for Siglecs. However, the role of tandem inner epitope for Siglec interaction has never been reported. Herein, we report the effect of internal glycan (by length and type) on the binding affinity and describe a simple and efficient chemo-enzymatic sugar nucleotide regeneration protocol for the preparative-scale synthesis of oligo-LacNAcs by the sequential use of ß1,4-galactosyltransferase (ß4GalT) and ß1,3-N-acetylglucosyl transferase (ß3GlcNAcT). Further modification of these oligo-LacNAcs was performed in one-pot enzymatic synthesis to yield sialylated and/or fucosylated analogs. A glycan library of 23 different sialosides containing various LacNAc lengths or Lac core with natural/unnatural sialylation and/or fucosylation was synthesized. These glycans were used to fabricate a glycan microarray that was utilized to screen glycan binding preferences against five different Siglecs (2, 7, 9, 14 and 15).