Development of monoclonal antibodies against bovine mucin core 2 b6 N-acetylglucosaminyltransferase Cheng-Ming Li 13 , Nirmal Joshee 2 , Kenneth B. Adler 3 and Pi-Wan Cheng 1,2 * 1 Departments of Pediatrics and Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 2 Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 3 Department of Anatomy, Physiological Sciences & Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC Molecular cloning techniques have been used to produce abundant amounts of recombinant glycosyltransferases for biochemical studies. We recently cloned a cDNA which encoded bovine mucin core 2 b6N-acetylglucosaminyl transferase (C2TF). Poly-histidine-C2TF fusion protein was generated from the cloned cDNA in the E. coli Xpress system and used to produce monoclonal antibodies (MAbs). We obtained seven hybridomas which secreted MAbs against bovine C2TF in mouse ascites with titers ranging from 1:1280 to 1:40960 as assessed by immunofluorescence assay (IF). Isotyping revealed that all seven MAbs were IgG (4 IgG1, 2 IgG2b and 1 IgG2a). The affinity constants (M 1 ) for these MAbs range from 5.4  10 7 to 1.2  10 9 . These MAbs recognized bovine C2TF in tissue sections and on Western blottings. Six of these MAbs reacted with human core 2-M enzyme and one with both core 2-L and core 2-M enzymes on Western blottings. Therefore, These antibodies should be useful for further study of bovine and human core 2 enzymes. Keywords: core 2 N-acetylglucosaminyltransferase, glycosyltransferase, monoclonal antibodies, mucin Introduction The oligosaccharides of glycoconjugates play crucial roles in many important biological processes, such as maintain- ing glycoproteins in circulation and regulating cell-cell in- teractions, differentiation, bacterial and viral adhesions, and protein solubility and stability [1,2]. In airway mucins, carbohydrate is the major constituent, which contributes to the rheological properties of the mucus gel that protects the respiratory tract from infection by airborne pathogens [3,4]. Mucin carbohydrates are very heterogeneous in size and structure, which are determined by sequential addition of different sugars as catalyzed by glycosyltransferases. N- Acetylglucosaminyltransferases belong to the group of gly- cosyltransferases which are responsible for elongation and branching of oligosaccharides [4–7]. The synthesis of branched mucin oligosaccharides, which include core 2, core 4, and blood group I structures [4–7], is determined solely by b6GlcNAc TF’s. In bovine tracheal epithelium, all three GlcNAc structures can be synthesized by one b6GlcNAc transferase [6] while in human myloid cells these structures are synthesized by three different enzymes [8–10]. The core 2 GlcNAc transferase, which catalyzes the transfers of GlcNAc from UDP-GlcNAc to Galb1-3Gal- NAc forming Galb1-3(GlcNAcb1-6) GalNAc structure, is one of the better characterized b6GlcNAc transferases. In competition with sialyltransferase (STGalNAc I/II), which catalyzes the transfer of NeuAc from CMP-NeuAc to Galb3GalNAc forming Galb3(NeuAca2-6)GalNAc struc- ture, C2TF is responsible for the elongation of mucin oli- gosaccharide from the C-6 of GalNAc. This concept has been elegantly demonstrated by Piller et al. [11] in the example of human T-cell activation, in which they showed that increase in mucin oligosaccharide chain length follow- ing activation of T-cells correlated with an increase in C2TF activity and a concomitant decrease in STGalNAc I/II ac- tivity [11]. Transfection of CHO cells [12] and human pan- creatic cancer cells [13], which do not express core 2 enzyme, with C2TF cDNA confirms the important role of this enzyme in the elongation of mucin-type glycans. In- creased C2TF activity has also been shown to be associated Glycoconjugate Journal 16, 555–562 (1999) © 2000 Kluwer Academic Publishers. Manufactured in The Netherlands *To whom correspondence should be addressed: Dr. Pi-Wan Cheng, Department of Biochemistry and Molecular Biology, University of Ne- braska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Tel.: (402) 559-5776; Fax: (402) 559-6650; E-mail: pcheng@unmc.edu