Seon-Joo Yoon's research while affiliated with Pacific Northwest Research Institute and other places

High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between "Os Fr.B" having 5-6 GlcNAc termini, vs. absence of such homotypic CCI between "Os Fr.1" having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.

The glycosyl epitope dimeric Lea (Lea-on-Lea), defined by mouse monoclonal antibody NCC-ST-421, was identified previously as tumor-associated antigen, expressed highly in various human cancer tissues and cell lines derived therefrom, but with minimal expression in various normal tissues. In the present study, we observed clearly higher expression of this epitope, defined by ST421, in beta-haptoglobin (beta-Hap) from sera of patients with colorectal cancer, compared to normal, healthy subjects or patients with chronic inflammatory processes (Crohn's disease, ulcerative colitis). We focused, therefore, on biochemical characterization of glycosyl epitope status expressed in beta-Hap. We concluded that the dimeric Lea epitope is carried by O-linked but not by N-linked structure, based on the following observations: i) Treatment of beta-Hap with alpha-L-fucosidase reduced its reactivity with ST421, but did not affect its reactivity with anti-Hap antibody. In contrast, treatment of purified beta-Hap with PNGase F, which releases N-linked glycans, had no effect on reactivity with ST421, but changed molecular mass from 40 kDa to 30 kDa. ii) Strong reactivity of Colo205 supernatant with ST421 was reduced clearly by pre-incubation of cells with benzyl-alpha-GalNAc.

N-glycosylation status of purified beta-haptoglobin from sera of 17 patients, and from sera of 14 healthy volunteer subjects, was compared by blotting with various lectins and antibodies. Patients in this study were diagnosed as having colon cancer through histological examination of each tumor tissue by biopsy. Blotting index of serum beta-haptoglobin with Aleuria aurantia lectin (AAL) was clearly higher for cancer patients than for healthy subjects. No such distinction was observed for blotting with three other lectins and two monoclonal antibodies. To determine tumor-associated reactivity of AAL binding as compared to inflammatory processes in colonic tissues, beta-haptoglobin separated from sera of 5 patients with Crohn's disease (CD), and 4 patients with ulcerative colitis (UC), was studied. All these cases, except one case of UC, showed AAL index lower than that in cancer cases, similarly to healthy subjects. The higher AAL binding of beta-haptoglobin in colon cancer patients than in healthy subjects appeared to be due to alpha-L-fucosyl residue, since it was eliminated by bovine kidney alpha-fucosidase treatment. N-linked glycans of serum haptoglobin from colon cancer patients vs. healthy subjects were released by N-glycanase, fluorescence-labeled, and subjected to normal-phase high performance liquid chromatography (NP-HPLC). Glycan structures were determined based on glucose unit (GU) values and their changes upon sequential treatment with various exoglycosidases. Glycosyl sequences and their branching status of glycans from 14 cases of serum beta-haptoglobin were characterized. The identified glycans were sialylated or nonsialylated, bi-antennary or tri-antennary structures, with or without terminal fucosylation.

N-glycosylation status of purified beta-haptoglobin separated from sera of patients with prostate cancer was studied in comparison to that of sera from patients with benign prostate diseases, or normal subjects. Two different approaches, as summarized below, one based on binding of lectins and antibodies to beta-haptoglobin, the other on mass spectrometry of released N-linked glycans from beta-haptoglobin, were performed. Some of the results were useful for distinction of prostate cancer vs. benign prostate diseases. i) Binding of Phaseolus vulgaris-L lectin (PHA-L), defining the GlcNAcbeta6Manalpha6Man side chain present in tri- or tetra-antennary N-linked glycans, to beta-haptoglobin was higher for cases of prostate cancer and high-grade prostate intraepithelial neoplasia than for benign diseases. Binding of Aleuria aurantia lectin (AAL) defining Fucalpha3-, alpha4-, or alpha6-GlcNAc, or monoclonal antibody directed to sialyl-Le(x), to beta-haptoglobin was also higher for some of the cancer cases than for benign diseases. Many other lectins and antibodies showed no binding to beta-haptoglobin, or showed no significant difference between cancer vs. benign diseases. ii) Mass spectrometric analysis of N-linked glycans of beta-haptoglobin released by Peptide N-glycosidase-F showed enhanced expression of monosialyl tri-antennary structures in prostate cancer cases. Thus, binding of PHA-L to affinity-purified beta-haptoglobin from sera of patients could lead to development of useful tools for differential diagnosis of prostate cancer vs. benign prostate diseases.

Growth of epidermoid carcinoma cell lines, A431 and KB, has been known to be controlled by the interaction of epidermal growth factor (EGF) and its receptor (EGFR) with tyrosine kinase. Ganglioside GM3 was previously found to interact with EGFR and to inhibit EGFR tyrosine kinase. However, motility of these cells, controlled by EGFR and ganglioside, was not studied. The present study is focused on the control mechanism of the motility of these cells through interaction of ganglioside, tetraspanin (TSP), and EGFR. Key results are as follows: (i) The level of EGFR expressed in A431 cells is ∼6 times higher than that expressed in KB cells, and motility of A431 cells is also much higher than that of KB cells, yet growth of A431 cells is either not affected or is inhibited by EGF. In contrast, growth of KB cells is enhanced by EGF. (ii) Levels of TSPs (CD9, CD82, and CD81) expressed in A431 cells are much higher than those expressed in KB cells, and TSPs expressed in A431 cells are reduced by treatment of cells with EtDO-P4, which inhibits the synthesis of glycosphingolipids (GSLs) and gangliosides. (iii) These TSPs are co-immunoprecipitated with EGFR in both A431 and KB cells, indicating that TSPs are closely associated with EGFR. (iv) High motility of A431 cells is greatly reduced, while low motility of KB cells is not affected, by treatment of cells with EtDO-P4. These results, taken together, suggest that there is a close correlation between high motility of A431 cells and high expression of EGFR and TSPs, and between ganglioside GM3/GM2 and TSP. A similar correlation was suggested between the low motility of KB cells and low levels of EGFR and TSP. The correlation between high motility and high level of EGFR with the ganglioside-TSP complex in A431 cells is unique. This is in contrast to our previous studies that indicate that motility of many types of tumor cells is inhibited by a high level of CD9 or CD82, together with growth factor receptors and integrins.

Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and beta-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in alpha-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIB-knocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR.

Water-soluble fraction (WSF) from edible mushroom hinmogi (Tremella fuciformis) were obtained by water extraction, and polysaccharides in the WSF were separated by ethanol precipitation. The inhibitory effects of the polysaccharides on 3T3-L1 adipocyte differentiation were evaluated by the reduction of peroxisome proliferators-activated receptor gamma (PPAR gamma) translation, triglyceride accumulation, Oil Red-O staining, and expression levels of PPAR gamma, CCAAT/ enhancer binding protein alpha (C/EBP alpha), and leptin. The PPAR gamma translation in 3T3-L1 cells was inhibited by the treatment with polysaccharide precipitated by 80% ethanol (P80) which showed highest inhibitory activity among polysaccharides tested. In addition, treatment of P80 to 3T3-L1 cells significantly inhibited the triglyceride accumulation, Oil Red-O staining, and mRNA expression of PPAR gamma, C/EBP alpha, and leptin in a dose-dependent manner. Based upon these results, P80 from edible mushroom hinmogi shows the inhibitory activity on the differentiation of 3T3-L1 adipocytes. Therefore, it might be employed as a potential anti-obesity material.

The ideal water-soluble dietary fiber for the fiber-enrichment of foods must be very low in viscosity, tasteless, odorless, and should produce clear solutions in beverages. Partially hydrolyzed guar gum (PHGG) produced from guar gum by enzymatic process has the same chemical structure with intact guar gum but less than one-tenth the original molecular length of guar gum, which make available to be used as film former, foam stabilizer and swelling agent. The viscosity of PHGG is about 10 mPa.s in 5% aqueous solution, whereas 1% solution of guar gum shows range from 2,000 to 3,000 mPa.s. In addition, PHGG is greatly stable against low pH, heat, acid and digestive enzyme. For these reasons, PHGG seems to be one of the most beneficial dietary fiber materials. It also showed that interesting physiological functions still fully exert the nutritional function of a dietary fiber. PHGG has, therefore, been used primarily for a nutritional purpose and became fully integrated food material without altering the rheology, taste, texture and color of final products. PHGG named as Benefiber(R) in USA has self-affirmation on GRAS status of standard grade PHGG. PHGG named as Sunfiber(R) is now being used in various beverages, food products and medicinal foods as a safe, natural and functional dietary fiber in all over the world.