Asparagine-linked Carbohydrate Research Articles

Analysis of Immunogenic Galactose-α-1,3-galactose-Containing N-Glycans in Beef, Mutton, and Pork Tenderloin by Combining Matrix-Assisted Laser Desorption/Ionization-Mass Spectroscopy and Capillary Electrophoresis Hyphenated with Mass Spectrometry via Electrospray Ionization.

Severe allergic reactions to certain types of meat following tick bites have been reported in geographic regions which are endemic with ticks. This immune response is directed to a carbohydrate antigen (galactose-α-1,3-galactose or α-Gal), which is present in glycoproteins of mammalian meats. At the moment, asparagine-linked complex carbohydrates (N-glycans) with α-Gal motifs in meat glycoproteins and in which cell types or tissue morphologies these α-Gal moieties are present in mammalian meats are still unclear. In this study, we analyzed α-Gal-containing N-glycans in beef, mutton, and pork tenderloin and provided for the first time the spatial distribution of these types of N-glycans in various meat samples. Terminal α-Gal-modified N-glycans were found to be highly abundant in all analyzed samples (55, 45, and 36% of N-glycome in beef, mutton, and pork, respectively). Visualizations of the N-glycans with α-Gal modification revealed that this motif was mainly present in the fibroconnective tissue. To conclude, this study contributes to a better understanding of the glycosylation biology of meat samples and provides guidance for processed meat products, in which only meat fibers are required as an ingredient (i.e., sausages or canned meat).

Knockout of the CMP-Sialic Acid Transporter SLC35A1 in Human Cell Lines Increases Transduction Efficiency of Adeno-Associated Virus 9: Implications for Gene Therapy Potency Assays.

Recombinant adeno-associated viruses (AAV) have emerged as an important tool for gene therapy for human diseases. A prerequisite for clinical approval is an in vitro potency assay that can measure the transduction efficiency of each virus lot produced. The AAV serotypes are typical for gene therapy bind to different cell surface structures. The binding of AAV9 on the surface is mediated by terminal galactose residues present in the asparagine-linked carbohydrates in glycoproteins. However, such terminal galactose residues are rare in cultured cells. They are masked by sialic acid residues, which is an obstacle for the infection of many cell lines with AAV9 and the respective potency assays. The sialic acid residues can be removed by enzymatic digestion or chemical treatment. Still, such treatments are not practical for AAV9 potency assays since they may be difficult to standardize. In this study, we generated human cell lines (HEK293T and HeLa) that become permissive for AAV9 transduction after a knockout of the CMP–sialic acid transporter SLC35A1. Using the human aspartylglucosaminidase (AGA) gene, we show that these cell lines can be used as a model system for establishing potency assays for AAV9-based gene therapy approaches for human diseases.

A synopsis of recent developments defining how N-glycosylation impacts immunoglobulin G structure and function.

Therapeutic monoclonal antibodies (mAbs) are the fastest growing group of drugs with 11 new antibodies or antibody-drug conjugates approved by the Food and Drug Administration in 2018. Many mAbs require effector function for efficacy, including antibody-dependent cell-mediated cytotoxicity triggered following contact of an immunoglobulin G (IgG)-coated particle with activating crystallizable fragment (Fc) γ receptors (FcγRs) expressed by leukocytes. Interactions between IgG1 and the FcγRs require post-translational modification of the Fc with an asparagine-linked carbohydrate (N-glycan). Though the structure of IgG1 Fc and the role of Fc N-glycan composition on disease were known for decades, the underlying mechanism of how the N-glycan affected FcγR binding was not defined until recently. This review will describe the current understanding of how N-glycosylation impacts the structure and function of the IgG1 Fc and describe new techniques that are poised to provide the next critical breakthroughs.

A single amino acid distorts the Fc γ receptor IIIb/CD16b structure upon binding immunoglobulin G1 and reduces affinity relative to CD16a

Therapeutic mAbs engage Fc γ receptor III (CD16) to elicit a protective cell-mediated response and destroy the target tissue. Newer drugs designed to bind CD16a with increased affinity surprisingly also elicit protective CD16b-mediated responses. However, it is unclear why IgG binds CD16a with more than 10-fold higher affinity than CD16b even though these receptors share more than 97% identity. Here we identified one residue, Gly-129, that contributes to the greater IgG binding affinity of CD16a. The CD16b variant D129G bound IgG1 Fc with 2-fold higher affinity than CD16a and with 90-fold higher affinity than the WT. Conversely, the binding affinity of CD16a-G129D was decreased 128-fold relative to WT CD16a and comparably to that of WT CD16b. The interaction of IgG1 Fc with CD16a, but not with CD16b, is known to be sensitive to the composition of the asparagine-linked carbohydrates (N-glycans) attached to the receptor. CD16a and CD16b-D129G displaying minimally processed oligomannose N-glycans bound to IgG1 Fc with about 5.2-fold increased affinity compared with variants with highly processed complex-type N-glycans. CD16b and the CD16a-G129D variant exhibited a smaller 1.9-fold affinity increase with oligomannose N-glycans. A model of glycosylated CD16b bound to IgG1 Fc determined to 2.2 Å resolution combined with a 250-ns all-atom molecular dynamics simulation showed that the larger Asp-129 residue deformed the Fc-binding surface. These results reveal how Asp-129 in CD16b affects its binding affinity for IgG1 Fc and suggest that antibodies engineered to engage CD16b with high affinity must accommodate the Asp-129 side chain.

Development of a colorimetric PNGase activity assay

PNGases are crucial targets and valuable tools in analyzing asparagine-linked carbohydrate moieties (N-glycans) of glycoproteins. Activity tests of PNGases have been little improved since their discovery four decades ago, and still rely on observing deglycosylation patterns of glycoproteins or glycopeptides using SDS-PAGE or HPLC analysis. These techniques cannot be easily adapted for automated sampling and high-throughput procedures. Herein, we describe a PNGase activity assay which relies on the conversion of WST-1, a yellowish, water-soluble tetrazolium dye (sodium 2-(4-Iodophenyl)-3-(4-nitro-phenyl)-5-(2,4-disulfophenyl)-2H-tetrazolate), into a blue formazan dye. In this work, we showed that WST-1 could be reduced by N-glycans, which were enzymatically released from glycoprotein substrates. After optimization of the assay conditions, the robustness of the method was challenged by quantifying the activity of various PNGase isoforms at different purification stages using a microwell plate reader. Furthermore, the assay could be used to obtain steady-state kinetics of PNGase H+ wild-type and mutant variants, which showed significant differences in their enzymatic reaction rates. The simplicity and robustness of this method might be of benefit for the detection of PNGase activity in routine applications of large amounts of samples.

CD16a with oligomannose-type N-glycans is the only “low-affinity” Fc γ receptor that binds the IgG crystallizable fragment with high affinity in vitro

Fc γ receptors (FcγRs) bind circulating IgG (IgG1) at the surface of leukocytes. Antibodies clustered at the surface of a targeted particle trigger a protective immune response through activating FcγRs. Three recent reports indicate that the composition of the asparagine-linked carbohydrate chains (N-glycans) of FcγRIIIa/CD16a impacted IgG1-binding affinity. Here we determined how N-glycan composition affected the affinity of the "low-affinity" FcγRs for six homogeneous IgG1 Fc N-glycoforms (G0, G0F, G2, G2F, A2G2, and A2G2F). Surprisingly, CD16a with oligomannose N-glycans bound to IgG1 Fc (A2G2) with a KD = 1.0 ± 0.1 nm This affinity represents a 51-fold increase over the affinity measured for CD16a with complex-type N-glycans (51 ± 8 nm) and is comparable with the affinity of FcγRI/CD64, the sole "high-affinity" FcγR. CD16a N-glycan composition accounted for increases in binding affinity for the other IgG1 Fc glycoforms tested (10-50-fold). This remarkable sensitivity could only be eliminated by preventing glycosylation at Asn162 with an Asn-to-Gln mutation; mutations at the four other N-glycosylation sites preserved tighter binding in the Man5 glycoform. None of the other low-affinity FcγRs showed more than a 3.1-fold increase upon modifying the receptor N-glycan composition, including CD16b, which differs from CD16a by only four amino acid residues. This result indicates that CD16a is unique among the low-affinity FcγRs, and modifying only the glycan composition of both the IgG1 Fc ligand and receptor provides a 400-fold range in affinities.

Site specificity of eel luteinizing hormone N-linked oligosaccharides in signal transduction

Eel luteinizing hormone (eelLH) is composed of a common α-subunit and hormone specific β-subunit, both of which contain asparagine-linked carbohydrate residues, located at positions 56 and 79 on the α-subunit and position 10 on the β-subunit. The specific roles of the individual carbohydrate chains are poorly defined in eel. Thus, we characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelLH β-subunit. Site-directed mutagenesis of the three N-linked glycosylation sites was performed to examine the function of individual glycosylation sites in secretion and signal transduction. The absence of the Asn79N-linked sugar chain slightly reduced secretion in Chinese hamster ovary (CHO) cells. The expression of eelLHβ/α (wild-type) in CHO suspension cells was increased by approximately 2-fold higher than that of attached CHO cells. By western blotting analysis, the molecular weight of wild-type was found to be 32 kDa. Mutants (β/α△56, β/α△79, and β△10/α) of the oligosaccharide chain at a single site showed molecular weights that were reduced by approximately 10%. However, the double mutant (β/α△56.79) molecular weight was decreased by more than 20% compared to the wild-type. Enzymatic digestion of oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants, including wild-type, were reduced to 25 kDa. The results obtained in the absence of carbohydrates at Asn56 of the α-subunit and at Asn10 of the β-subunit revealed their roles in signal transduction through the eelLH receptor. The EC50 value of the cAMP response at Asn79 of the α-subunit was increased by 5-fold, whereas the maximum response was dramatically reduced to 17.8% of wild-type levels. Specifically, removal of the carbohydrates at double mutant (β/α△56.79) is approximately 85% to wild-type levels in biopotency. These results revealed the site-specific roles of eelLH carbohydrate residues. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelLH receptor.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates (N-glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N-glycans (23%). These proportions indicated restricted N-glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N-glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N-glycoforms similar to NK cell-derived CD16a but yielded N-glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N-glycan composition affected antibody binding: CD16a with oligomannose N-glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N-glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N-glycan composition and antibody-binding affinity.

Carbohydrate–Polypeptide Contacts in the Antibody Receptor CD16A Identified through Solution NMR Spectroscopy

Asparagine-linked carbohydrates (N-glycans) are common modifications of eukaryotic proteins that confer multiple properties, including the essential stabilization of therapeutic monoclonal antibodies. Here we present a rapid and efficient strategy for identifying N-glycans that contact polypeptide residues and apply the method to profile the five N-glycans attached to the human antibody receptor CD16A (Fc γ receptor IIIA). Human embryonic kidney 293S cells expressed CD16A with 13CU-labeled N-glycans using standard protein expression techniques and medium supplemented with 3 g/L [13CU]glucose. Anomeric resonances on the protein-linked N-acetylglucosamine residue at the reducing end of the glycan are particularly well suited to studies of multiply glycosylated N-glycoproteins because only one reducing end and nitrogen-linked residue is present in each N-glycan. Correlations between anomeric 1H1 and 13C1 nuclei on the reducing end residue generate crosspeaks in a conventional two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) experiment that appear in a region of the spectrum devoid of other carbohydrate peaks or background protein signals. Two N-glycan peaks corresponding to the N45 and N162 N-glycans were dispersed from the rapidly averaged peaks corresponding to the N38, N74, and N169 N-glycans. We used a combination of NMR and 1 μs all-atom computational simulations to identify unexpected contacts between the N45 N-glycan and CD16A polypeptide residues.

Complex carbohydrates as a predictive marker in metastatic colorectal cancer (mCRC) cancer patients treated with a vascular endothelial growth factor (VEGF) inhibitor and chemotherapy.

e15134 Background: Aberrant expression of complex carbohydrates, known as glycans, is emerging as a key driver in oncopathology. Elevated levels of sialic acid sugars affect tumor growth, escape from apoptosis, metastasis formation, and resistance to therapy. Changes in sialylation can be associated with malignant transformation and with progression and poor prognosis of carcinomas, which can be explained by the recognition of malignant cells by selectins, causing interactions of tumor cells with platelets, leukocytes and endothelium facilitating metastasis. Secreted or proteolytically released carcinoma mucins bearing unusual forms of sialylation can be detected in the bloodstream and are used as diagnostic and prognostic aids. Bevacizumab directly binds VEGF (a glycoprotein) to inhibit angiogenesis. The aim of this study was to analyze the pool of asparagine-linked complex carbohydrates (i.e. the N-glycome), of whole serum to identify sugars that are associated with survival in mCRC patients treated with a VEGF and chemotherapy. Methods: We used our recently developed high-throughput automated platform to precisely measure the levels of complex serum carbohydrates in patients (n = 68) with metastatic colorectal cancer samples of patients who went on to receive bevacizumab and chemotherapy. Survival analysis was performed using the R package ‘survival’. Results: We observed several sialic-acid bearing glycans that correlated with overall survival and progression free survival. A complex tetra-antennary, highly sialylated glycan, known as A4G4S4, correlated with reduced overall survival probability (p = 0.0007, hazard ratio = 1.86). High levels of a sialylated tri-antennary glycan, called A3G3S3 demonstrated significantly reduced progression-free survival (p = 0.002). Conclusions: We believe that sialylated glycans could be excellent predictive markers of response to therapy and warrant further evaluation in clinical trials. Tri and tetra-antennary sialyated glycoproteins were associated with poorer survival in our patient cohort, suggesting that glycans could be key drivers of pathobiology.

Deciphering the Glycosylation Code

Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the “glycosylation code” that relates these effects to protein sequence remains unsolved. We report atomically detailed replica exchange molecular dynamics simulations in explicit solvent that systematically investigate the impact of glycosylation upon peptides with the central sequon Pro-Asn-Gly/Ala-Thr-Trp/Ala. These simulations suggest that the effects of glycosylation may be quite sensitive to steric crowding by the side chain immediately following the glycosylation site but less sensitive to stacking interactions with the aromatic Trp residue. In addition, we compare our simulated ensembles with the known structures for full length glycoproteins. These structures corroborate the simulations and also suggest a remarkable consistency between the intraprotein and protein-glycan interactions of natural glycoproteins. Moreover, our analysis highlights the significance of left-handed conformations for...

Deciphering the Glycosylation Code

Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the "glycosylation code" that relates these effects to protein sequence remains unsolved. We report atomically detailed replica exchange molecular dynamics simulations in explicit solvent that systematically investigate the impact of glycosylation upon peptides with the central sequon Pro-Asn-Gly/Ala-Thr-Trp/Ala. These simulations suggest that the effects of glycosylation may be quite sensitive to steric crowding by the side chain immediately following the glycosylation site but less sensitive to stacking interactions with the aromatic Trp residue. In addition, we compare our simulated ensembles with the known structures for full length glycoproteins. These structures corroborate the simulations and also suggest a remarkable consistency between the intraprotein and protein-glycan interactions of natural glycoproteins. Moreover, our analysis highlights the significance of left-handed conformations for compact β-hairpins at glycosylation sites. In summary, these studies elucidate basic biophysical principles for the glycosylation code.

Expression, purification and preliminary crystallographic analysis of Drosophila melanogaster lysosomal α-mannosidase.

The lysosomal α-mannosidases are class II mannosidases that belong to glycoside hydrolase family 38 and play an important role in the degradation of asparagine-linked carbohydrates of glycoproteins. Based on peptide similarity to human and bovine lysosomal mannosidase (LM), recombinant α-mannosidase from Drosophila melanogaster (dLM408) was cloned and heterologously expressed in Pichia pastoris. The recombinant form of dLM408 designed for structural analysis lacks the transmembrane domain and was crystallized using standard vapour-diffusion and counter-diffusion techniques. The crystals grew as flat plates and as tetragonal bipyramids, respectively. The plate-shaped crystals exhibited the symmetry of space group P2(1)2(1)2(1) and diffracted to a minimum d-spacing of 3.5 Å.

Enzymatic removal of asparagine-linked carbohydrate chains from heterodimer human chorionic gonadotrophin and effect on bioactivity

The native form of human chorionic gonadotropin (hCG) is a heterodimer protein with two asparagine (Asn)-linked carbohydrate chains on each subunit. Removal of the Asn-linked carbohydrate chains from hCG has resulted in hCG variants with consistent antagonistic properties on isolated murine cells. Specific and direct enzymatic removal of these carbohydrate chains from native hCG with resultant antagonistic properties has not been reported. An antagonist to the hCG/luteinising hormone (LH) receptor could be used as an anticancer therapy, emergency contraceptive or for therapeutic resolution of ectopic pregnancies. Therefore, our aim was to use enzymes to specifically remove Asn-linked carbohydrate chains from hCG in the heterodimer form and analyse the resultant bioactivity. Native hCG was treated with endoglycosidases, carbohydrate removal was analysed with electrophoresis and the hCG variants were tested for altered bioactivity with human and murine cells. Endoglycosidases were able to cleave most of the Asn-linked carbohydrate chains from the native hCG. The deglycosylated hCG demonstrated a 75% reduction in bioactivity on a murine Leydig cell line and a 65% reduction in bioactivity on human granulosa cells. These results exemplify a simple and efficient method for creating deglycosylated hCG and provide the most direct evidence for the importance of Asn-linked carbohydrate chains in maintaining hCG bioactivity.

The N‐glycans of yellow jacket venom hyaluronidases and the protein sequence of its major isoform in Vespula vulgaris

Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.

Characterisation of the biosynthesis and processing of the neutrophil granule membrane protein CD63 in myeloid cells.

The biosynthesis and processing of the neutrophil granule membrane protein CD63, present in azurophil granules, was investigated in four myeloid cell lines. The amount of CD63 synthesised differed, so did the amount of protein processed to high molecular weight forms, with the demonstration of a more prominent synthesis of CD63 in K562 cells. Newly synthesised CD63 was initially detected as two precursor forms with molecular weight of 32 and 35 kDa, respectively. These two initial forms were processed further to yield high molecular weight forms of CD63 with a mean molecular weight of 50 kDa. Treatment with endoglycosidase H or N-glycosidase F revealed a protein core, free from asparagine-linked carbohydrates, with a molecular weight of 23 kDa. Newly synthesised CD63 was susceptible to digestion with endoglycosidase H, and the protein was not completely resistant to endoglycosidase H until after 4 h of chase, indicating that transport through the medial and trans-Golgi complex with conversion of high-mannose carbohydrates to complex oligosaccharide side chains had occurred. This finding indicates a relatively long processing time for CD63 compared to that of soluble azurophil granule proteins. By digestion with O-glycanase, the existence of O-linked oligosaccharides on CD63 could not be demonstrated. Biosynthetic labelling of cells in the presence of brefeldin A showed the importance of a functional Golgi apparatus for processing of the protein to its high molecular weight forms.

Germ Cell Survival Through Carbohydrate-Mediated Interaction with Sertoli Cells

Spermatogenesis is a precisely regulated process in which the germ cells closely interact with Sertoli cells. The molecular basis of this cell-cell adhesion is unknown. Here, we demonstrate that targeted disruption of Man2a2, a gene encoding alpha-mannosidase IIx (MX), an enzyme that forms intermediate asparagine-linked carbohydrates (N-glycans), results in Man2a2 null males that are largely infertile. The Man2a2 null spermatogenic cells fail to adhere to Sertoli cells and are prematurely released from the testis to epididymis. We identified an N-glycan structure that plays a key role in germ cell-Sertoli cell adhesion and showed that a specific carbohydrate was required for spermatogenesis.

Purification and Characterization of Recombinant Human Lysosomal α-Mannosidase

Lysosomal α-mannosidase (EC 3.2.1.24) is required in the degradation of the asparagine-linked carbohydrates of glycoproteins. Deficiency of this enzyme leads to the lysosomal storage disorder α-mannosidosis. As an initial step toward enzyme replacement therapy for α-mannosidosis, the human lysosomal α-mannosidase cDNA was cloned into the pcDNA 3.1 vector and expressed in Chinese hamster ovary cells. Dimethyl sulfoxide (DMSO) added to the cell culture media to induce growth arrest led to a 4-fold increase in the enzyme production, with an average yield of 3.2 mg L−1 day−1. α-Mannosidase was secreted as an active homodimer of a 130-kDa precursor that was proteolyzed into two polypeptides of 55 and 72 kDa during the subsequent purification of the enzyme. N-terminal sequence analysis of the purified enzyme revealed that the proteolysis occurred close to a cleavage site previously identified in the intracellular form of lysosomal α-mannosidase. Generation of monoclonal antibodies against the recombinant enzyme made it possible to develop a single-step immunoaffinity purification procedure for α-mannosidase. The immunoaffinity-purified enzyme which mainly consisted of the 130-kDa precursor, displayed specific activity and kinetics similar to those of the processed form. Recombinant α-mannosidase was taken up by cultured α-mannosidosis fibroblasts and was trafficked to the lysosomes via the mannose 6-phosphate pathway where it reduced the amounts of stored mannose-containing oligosaccharides.

The man/GalNAc-4-SO4-receptor has multiple specificities and functions.

Carbohydrate-specific recognition by receptors contributes to a variety of different biological processes. These include: the folding and assembly of glycoproteins in the endoplasmic reticulum (Helenius 1994; Williams 1995; Trombetta and Helenius 1998), transport of glycoproteins to lysosomes (Kornfeld 1990, 1992), control of the circulatory half-life of individual glycoproteins (Drickamer 1991; Baenziger et al. 1992; Drickamer and Taylor 1993), endocytosis (Ashwell and Harford 1982; Drickamer and Taylor 1993), phagocytosis (Stahl and Ezekowitz 1998), cellular recognition during coagulation (McEver and Cummings 1997; Rosenerg et al. 1997), inflammation (Rosen and Bertozzi 1994; McEver et al. 1995; Lowe 1997), and development (Ioffe and Stanley 1994; Metzler et al. 1994; Chui et al. 1997). We recently described the first example of a carbohydrate-specific receptor, the Man/Ga1NAc-4-SO4 receptor (Fiete et al. 1998), that utilizes two distinct regions with different structural motifs to bind completely different carbohydrate structures. Furthermore, different cell types, such as macrophages and hepatic endothelial cells, express forms of the Man/Ga1NAc-4-SO4 receptor that differ with respect to their carbohydrate specificity. Macrophages express a mannose (Man) specific form of the receptor (Man-receptor), while hepatic endothelial cells express an N-acetylgalactosamine-4-SO4 (Ga1NAc-4-SO4) specific form of the receptor (Ga1NAc-4-SO4-receptor; Fiete and Baenziger 1997; Fiete et al. 1997). The regulated expression of different specificities by the Man/GalNAc-4-SO4-receptor indicates that the Man-receptor and the Ga1NAc-4-SO4receptor have different biological functions. Asparagine-linked carbohydrate structures terminating with the sequence SO4-4Ga1NAcβ1,4G1cNAcβ1,2Manα (S4GGnM) are present on highly restricted populations of glycoproteins, including the pituitary glycoprotein hormones LH and TSH (Green and Baenziger 1988a,b; Baenziger and Green 1991; Stockell Hartree and Renwick 1992; Thotakura and Blithe 1995). The synthesis of these sulfated oligosaccharides is protein specific, and is both hormonally and developmentally regulated (S.M. Dharmesh and J.U. Baenziger, unpubl. obs.; Dharmesh and Baenziger 1993; Manzella et al. 1997). Further, recognition of terminal Ga1NAc-4-SO4 on the oligosaccharides of LH by the Ga1NAc-4-SO4-receptor residing in hepatic endothelial cells (Fiete et al. 1991) plays a critical role in vivo by controlling the circulatory half-life of LH at key points during the ovulatory cycle (Baenziger et al. 1992; Baenziger 1996; Manzella et al. 1996). In contrast, recognition of carbohydrates terminating with Man, Fucose (Fuc), or N-acetylglucosamine (G1cNAc) by the Man-receptor, present on terminally differentiated macrophages, is thought to be essential for phagocytosis of pathogens such as yeasts, bacteria, and viruses (Fraser et al. 1998; Stahl and Ezekowitz 1998). Thus, the Man/Ga1NAc-4-SO4-receptor, in the form of the Man-receptor or the GalNAc-4-SO4-receptor, may fulfill distinct roles that reflect its ligand specificity as well as the setting in which it is expressed.

Thermodynamic Binding Studies of Lectins from the Diocleinae Subtribe to Deoxy Analogs of the Core Trimannoside of Asparagine-linked Oligosaccharides

Lectins from seven different species of the Diocleinae subtribe have been recently isolated and characterized in terms of their carbohydrate binding specificities (Dam, T. K., Cavada, B. S., Grangeiro, T. B., Santos, C. F., de Sousa, F. A. M., Oscarson, S., and Brewer, C. F. (1998) J. Biol. Chem. 273, 12082-12088). The lectins included those from Canavalia brasiliensis, Cratylia floribunda, Dioclea rostrata, Dioclea virgata, Dioclea violacea, and Dioclea guianensis. All of the lectins exhibited specificity for Man and Glc residues, but much higher affinities for the branched chain trimannoside, 3,6-di-O-(alpha-d-mannopyranosyl)-d-mannose, which is found in the core region of all asparagine-linked carbohydrates. In the present study, isothermal titration microcalorimetry is used to determine the binding thermodynamics of the above lectins, including a new lectin from Canavalia grandiflora, to a complete series of monodeoxy analogs of the core trimannoside. From losses in the affinity constants and enthalpies of binding of certain deoxy analogs, assignments are made of the hydroxyl epitopes on the trimannoside that are involved in binding to the lectins. The pattern of binding of the deoxy analogs is similar for all seven lectins, and similar to that of concanavalin A which is also a member of the Diocleinae subtribe. However, differences in the magnitude of the thermodynamic binding parameters of the lectins are observed, even though the lectins possess conserved contact residues in many cases, and highly conserved primary sequences. The results indicate that non-contact residues in the lectins, even those distant from the binding sites, modulate their thermodynamic binding parameters.