Production and characterization of monoclonal antibodies specific to lactotriaosylceramide

Abstract

We have established hybridoma cell lines producing monoclonal antibodies (mAbs) directed to N-acetylglucosaminylβ1-3galactose (GlcNAcβ1-3Gal) residue by immunizing BALB/c mice with lactotriaosylceramide (Lc(3)Cer). These obtained hybridoma cells, specific to Lc(3)Cer, were dual immunoglobulin (Ig)-producing cells which secreted both IgM and IgG molecules as antibodies. The established mAbs are able to react with not only Lc(3)Cer but also GlcNAcβ1-3-terminal glycosphingolipids (GSLs) despite branching or lactosamine chain lengths and human transferrin with terminal GlcNAc residues. Comparison of the variable regions of the cloned IgM and IgG by reversed transcription-polymerase chain reaction analysis confirmed that the variable regions determine the specificity, the other amino acids are conserved, and these mAbs are encoded by J558 and Vκ-21family genes. Furthermore, we have analyzed the expression of GSLs with GlcNAcβ1-3 epitope in acute leukemia cell lines and mouse fetal tissues using these mAbs, in which antigens were distributed comparatively. These mAbs are useful for studying the precise distribution of GlcNAcβ1-3Gal-terminating GSL expression in tissues as well as for detecting GSLs carrying terminal GlcNAcβ1-3Gal carbohydrate structure.

MiR‐30a‐5p inhibits proliferation and metastasis of hydatidiform mole by regulating B3GNT5 through ERK/AKT pathways

“…40 Specifically, Lc3 was shown to be elevated on the cell surface of human pro-myelocytic leukaemia HL60 cells. 41 Additionally, the expression of B3GNT5 was revealed to be markedly decreased in the high-grade as a tumour-promoting gene. 21 Our results suggest that miR-30a may affect the development of HM by directly regulating B3GNT5.…”

“…This enzyme, together with its associated glycosidic product (Lc3), plays a role in human malignant diseases, embryonic development and cell differentiation 40 . Specifically, Lc3 was shown to be elevated on the cell surface of human pro‐myelocytic leukaemia HL60 cells 41 . Additionally, the expression of B3GNT5 was revealed to be markedly decreased in the high‐grade ovarian cancer cell line A2780; however, it was increased in its cisplatin‐resistant subline A2780‐cp 42 .…”

Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

“…This enzyme, together with its associated glycosidic product (Lc3), plays a role in human malignant diseases, embryonic development and cell differentiation. Specifically, Lc3 was shown to be elevated on the cell surface of human pro-myelocytic leukemia HL60 cells67. Moreover, it has also been suggested as a differentiation-associated GSL in the bone marrow of acute myeloid leukemia patients with corresponding elevated B3GNT5 expression8.…”

Inhibition of Rab prenylation by statins induces cellular glycosphingolipid remodeling

“…We noticed on immunoblots that a polar, anti-Lc3Cer reactive GSL was concurrently expressed in statin-treated A431 cells. We suspected that this species may be nLc5, which is also a product of Lc3Cer synthase, has the same terminal GlcNAcβ(1-3) Gal as Lc3Cer and is recognized by anti-Lc3Cer MAC-1 (Togayachi et al 2001;Nozaki et al 2010). To confirm this, and identify additional Lc3Cer-related slow-migrating complex GSLs, a total neutral GSL fraction was prepared from A431G cells and probed by TLC immunoblot with anti-Lc3Cer (MAC-1), anti-nLc4 (FE-A5) or anti-SSEA1 (MC-480) (Figure 2D).…”

“…Mouse anti-Lc3Cer (clone MAC-1) was a generous gift of Dr Yasunori Kushi, Nihon University, Tokyo (Nozaki et al 2010). Mouse anti-GM3, clone DH2 was generously provided by Sen-itiroh Halkomori (Pacific Northwest Research Laboratory, Seattle, WA) (Dohi et al 1988).…”