MUCIN BIOSYNTHESIS - MOLECULAR-CLONING AND EXPRESSION OF BOVINE LUNG MUCIN CORE-2 N-ACETYLGLUCOSAMINYLTRANSFERASE CDNA

A cDNA clone containing a 2,150-bp insert was isolated from a bovine l ung lambda gt10 cDNA library by cross-species hybridization using a DN A probe generated by polymerase chain reaction (PCR) employing a human cDNA that encodes mucin core 2 beta 6-N-acetylglucosaminyltransferase (hC2TF) as the template. The bovine cDNA (bcDNA) insert was devoid of 220 bp of the 5' portion of the C2TF open reading frame (ORF), as pre dicted from the human counterpart. Southern blotting analysis suggeste d that the coding region of this C2TF gene is in one exon. To construc t a full-length bovine C2TF (bC2TF) cDNA, a genomic DNA fragment conta ining the 5' portion of the ORF of the bC2TF gene was cloned from a la mbda EMBL bovine genomic DNA library and ligated to the 5' end of the cloned cDNA insert. DNA sequence analysis showed that the complete ORF of bC2TF gene was 1,281 bp in length, which corresponds to a polypept ide of 427 amino acids. Catalytically active bC2TF was expressed in sf 21 insect cells infected with recombinant baculovirus containing the O RF of the bC2TF gene. The recombinant bC2TF catalyzed the synthesis of core 2, but not core 4 and blood group I structures. Western blotting analysis showed that the recombinant bC2TF migrated with the same mob ility (similar to 55 kD) as the native bovine tracheal C2TF. Immunohis tochemical analysis showed that in bovine trachea, the bC2TF was prese nt at the surface epithelium and in the submucosal glands, with the la tter being the major site of distribution.