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Am. J. Respir. Cell Mol. Biol., Volume 18, Number 3, March, 1998 343-352

Mucin Biosynthesis: Molecular Cloning and Expression of Bovine Lung Mucin Core 2 N-Acetylglucosaminyltransferase cDNA

Cheng-Ming Li, Kenneth B. Adler, and Pi-Wan Cheng

Departments of Pediatrics, Biochemistry, and Biophysics, University of North Carolina, Chapel Hill, North Carolina; and Departments of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

A cDNA clone containing a 2,150-bp insert was isolated from a bovine lung lambda gt10 cDNA library by cross-species hybridization using a DNA 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 predicted from the human counterpart. Southern blotting analysis suggested that the coding region of this C2TF gene is in one exon. To construct a full-length bovine C2TF (bC2TF) cDNA, a genomic DNA fragment containing the 5' portion of the ORF of the bC2TF gene was cloned from a lambda 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 polypeptide of 427 amino acids. Catalytically active bC2TF was expressed in sf21 insect cells infected with recombinant baculovirus containing the ORF 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 mobility (~ 55 kD) as the native bovine tracheal C2TF. Immunohistochemical analysis showed that in bovine trachea, the bC2TF was present at the surface epithelium and in the submucosal glands, with the latter being the major site of distribution.




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