Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve

Yamawaki, Masanaga; Ariga, Toshio; Bigbee, John W.; Ozawa, Hideki; Kawashima, Ichiro; Tai, Tadashi; Kanda, Takashi; Yu, Robert K.

doi:10.1002/(sici)1097-4547(19960615)44:6<586::aid-jnr9>3.0.co;2-7

Cited by 16 publications

(13 citation statements)

References 43 publications

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“…This result clearly demonstrates that the neurosphere-forming cells prepared in this study are NSCs as reported previously (11). To detect glycoproteins carrying HNK-1 in NSCs, we analyzed cell lysates prepared from these NSCs by Western blotting using NGR50, an anti-HNK-1 mouse IgG antibody (25). One major protein band reactive with NGR50, corresponding to an apparent molecular mass of 280 kDa (Fig.…”

Section: Resultssupporting

confidence: 87%

“…Materials-NGR50 mouse monoclonal antibody (IgG), prepared from culture supernatants of an NGR50 hybridoma cell line, was used as an anti-HNK-1 antibody (25). Other antibodies used in this study are shown in supplemental Table 1.…”

Section: Methodsmentioning

confidence: 99%

“…Immunocytochemistry-NSCs prepared from neurospheres were plated onto chamber slides (Nalge Nunc International, Naperville, IL) coated with poly-L-ornithine (Sigma-Aldrich) and fibronectin (Sigma-Aldrich) and fixed in PBS containing 4% paraformaldehyde. The NSCs were treated for 2 h with PBS containing 3% fetal bovine serum and 0% or 0.1% Triton X-100 and then stained with primary antibodies such as Rat401 antinestin monoclonal antibody (BD Biosciences), AK97 anti-SSEA-1 monoclonal antibody (IgM) (27), anti-␤-III tubulin monoclonal antibody (Sigma-Aldrich), anti-glial fibrillary acidic protein polyclonal antibody (DAKO Cytomation, Glostrup, Denmark), anti-galactocerebroside monoclonal antibody (Millipore), MTn12 anti-TNC rat monoclonal antibody (Sigma-Aldrich), and NGR50 anti-HNK-1 monoclonal antibody (25) and secondary antibodies such as anti-mouse IgG antibody conjugated with Alexa Fluor 488 (BD Biosciences), anti-mouse IgM antibody conjugated with Alexa Fluor 488, anti-rabbit IgG antibody conjugated with Alexa Fluor 488, antirat IgG antibody conjugated with DyLight488 (Jackson ImmunoResearch, West Grove, PA), and anti-mouse-IgG antibody conjugated with Cy3 (Jackson ImmunoResearch) (supplemental Table 1). Nuclei were stained with 2 g/ml Hoechst 33258 (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

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HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Yagi

Yanagisawa

Suzuki

et al. 2010

Journal of Biological Chemistry

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Neural stem cells (NSCs) possess high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into neuronal and glial cells. NSCs are the source for neurogenesis during central nervous system development from fetal and adult stages. Although the human natural killer-1 (HNK-1) carbohydrate epitope is expressed predominantly in the nervous system and involved in intercellular adhesion, cell migration, and synaptic plasticity, the expression patterns and functional roles of HNK-1-containing glycoconjugates in NSCs have not been fully recognized. We found that HNK-1 was expressed in embryonic mouse NSCs and that this expression was lost during the process of differentiation. Based on proteomics analysis, it was revealed that the HNK-1 epitopes were almost exclusively displayed on an extracellular matrix protein, tenascin-C (TNC), in the mouse embryonic NSCs. Furthermore, the HNK-1 epitope was found to be present only on the largest isoform of the TNC molecules. In addition, the expression of HNK-1 was dependent on expression of the largest TNC variant but not by enzymes involved in the biosynthesis of HNK-1. By knocking down HNK-1 sulfotransferase or TNC by small interfering RNA, we further demonstrated that HNK-1 on TNC was involved in the proliferation of NSCs via modulation of the expression level of the epidermal growth factor receptor. Our finding provides insights into the function of HNK-1 carbohydrate epitopes in NSCs to maintain stemness during neural development. Neural stem cells (NSCs)4 are undifferentiated neural cells characterized by their high proliferative potential and the capacity for self-renewal with retention of multipotency to differentiate into brain-forming cells, such as neurons, astrocytes, and oligodendrocytes (1-3). Environmental factors of NSCs, such as various growth factors, the extracellular matrix (ECM), and cell adhesion molecules, are known to play important roles in the maintenance of the stem cell population throughout specific cell lineage pathways (4 -7).Glycoconjugates, including glycoproteins, proteoglycans, and glycolipids, are expressed mainly on the cell surface as ECM, and they are known to regulate cell-to-cell communications. Certain glycoconjugates also serve as excellent biomarkers at various stages of the cellular differentiation of NSCs and play important functional roles in determining cell fate (8 -12). For example, stage-specific embryonic antigen-1 (SSEA-1), which is well known as a specific maker of undifferentiated cells including mouse embryonic stem cells, is expressed on NSCs and associated with cell migration (8, 10, 13). Recently, we have also demonstrated that cells positive for GD3 ganglioside (NeuAc␣2-8NeuAc␣2-3Gal␤1-4Glc␤1-1ЈCer) isolated from mouse brains of various ages possess characteristics of neural stem cells (11).The human natural killer-1 (HNK-1) carbohydrate epitope (CD57) was originally reported as a specific antigenic determinant for human natural killer cells (14) but is now widely known ...

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Section: Resultssupporting

confidence: 87%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Yagi

Yanagisawa

Suzuki

et al. 2010

Journal of Biological Chemistry

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“…The reactivity (titer) of the antiserum against a wide range of GSLs containing the same neutral oligosaccharide backbone, such as GM1, GA1, GD1b and GD1a was determined using conventional ELISA procedure (17)(18)(19) as modified in our laboratory. Briefly, wells of an ELISA titer plate were coated with 50 ll of the GSL (10 lg/ml) solution in ethanol.…”

Section: Determination Of the Antiserum Titer By Enzyme Linked Immunomentioning

confidence: 99%

Lack of Apparent Neurological Abnormalities in Rabbits Sensitized by Gangliosides

Dasgupta

2004

Neurochem Res

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Two very high titer polyclonal antibodies against two ganglioside antigens, GM1 and GD1a, have been raised in New Zealand white rabbits using a homogeneous suspension of the highly purified antigens in Keyhole Limpet Hemocyanin and Freund's adjuvant. The antisera were prepared over a period of 6 months with repeated injections of the ganglioside suspension, followed by an intravenous injection of the purified ganglioside solution, and collecting the serum (approximately 50 ml) at defined time intervals. The GM1-antibody, thus prepared, showed a cross reactivity toward GDlb and asialo-GM1 (GA1), while the GDla-antibody reacted with GD1a, GM1 and GA1 and GD1b as determined by immuno-overlay and ELISA methods. The titer for GM1 antiserum, determined by'ELISA, was greater than 1/10,000 dilution while the titer for GD1a antibody was greater than 1/5000 dilution. No neurological or behavioral abnormality was observed during the period of antiserum production. To evaluate any likely pathological damage caused by such a high titer ganglioside-antibody, autopsy of CNS as well PNS tissues from the rabbits were carried out after the final bleeding. No obvious pathological changes, including demyelination, were noted in any of the four rabbits. These observations cast doubt as to the direct effect of anti-ganglioside antibody induced neurological and pathological disorders.

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“…Anti-SGGL antibody activities were also detected by enzymelinked immunosorbent assay (ELISA) according to a procedure described previously [26]. Microtiter wells (Coming, Wexford, Pa., USA) were coated with 20 ng of test antigens for titer determination and 1-100 ng for the sensitivity study.…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Heterogeneity of antibody specificity in Taiwanese patients with polyneuropathy and IgM paraproteinemia

et al. 1998

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About half of the Caucasian patients with chronic polyneuropathy and IgM paraproteinemia show serum anti-myelin-associated glycoprotein (MAG) and anti-sulfoglucuronosyl glycosphingolipid (SGGLs) activities. These antibody activities have been demonstrated to react with a carbohydrate epitope known as the HNK-1 or sulfoglucuronic acid (SGA) epitope. However, in Asian populations the occurrence of serum anti-SGA activities has been reported to be relatively rare. We investigated 5 cases of chronic polyneuropathy with IgM paraproteinemia from Taiwan and found that 3 of them had high-titer serum anti-SGA (SGGL/MAG) antibody activities. The clinical symptoms of these 3 patients were consistent with sensory dominant polyneuropathy with a severer involvement of the lower limbs than of the upper limbs. Electromyography and nerve conduction studies revealed severe sensory nerve involvement (no response in 3 cases) and moderate slowing of motor conduction velocity (MCV) without conduction block. The decrease in MCV correlated well with anti-SGA antibody titer (less than 30 m/s with the titration of 1:12, 800, normal 55-60 m/s). Pathological findings showed active demyelinating polyneuropathy with myelin ovoid and myelinated fiber loss. Our data suggest that anti-SGGL antibody activities may not be very rare among Asian populations. Additionally, there seems an intriguing possibility that the titer of this antibody correlates with the severity of peripheral nerve involvement in patients of demyelinating polyneuropathy with IgM paraproteinemia.

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Generation and characterization of anti-sulfoglucuronosyl paragloboside monoclonal antibody NGR50 and its immunoreactivity with peripheral nerve

Cited by 16 publications

References 43 publications

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

HNK-1 Epitope-carrying Tenascin-C Spliced Variant Regulates the Proliferation of Mouse Embryonic Neural Stem Cells

Lack of Apparent Neurological Abnormalities in Rabbits Sensitized by Gangliosides

Heterogeneity of antibody specificity in Taiwanese patients with polyneuropathy and IgM paraproteinemia

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