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Infect Immun, February 1998, p. 741-746, Vol. 66, No. 2
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Identification of a Putative Precursor to the Major Surface Glycoprotein of Pneumocystis carinii

Susan M. Sunkin,¹ Michael J. Linke,^2,3 Francis X. McCormack,⁴ Peter D. Walzer,^2,3 and James R. Stringer^1,*

Department of Molecular Genetics, Biochemistry and Microbiology¹ and Divisions of Infectious Disease² and Pulmonary/Critical Care Medicine,⁴ Department of Internal Medicine, University of Cincinnati College of Medicine, and Cincinnati VA Medical Center,³ Cincinnati, Ohio 45267-0524

Received 12 August 1997/Returned for modification 8 October 1997/Accepted 1 December 1997

The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is a family of proteins encoded by a family of heterogeneous genes. Messenger RNAs encoding different MSGs each begin with the same 365-bp sequence, called the Upstream Conserved Sequence (UCS), which is in frame with the contiguous MSG sequence. The UCS contains several potential start sites for translation. To determine if translation of MSG mRNAs begins in the UCS, polyclonal antiserum was raised against the 123-amino-acid peptide encoded by the UCS. The anti-UCS serum reacted with a P. carinii protein that migrated at 170 kDa; however, it did not react with the mature MSG protein, which migrates at 116 kDa. A 170-kDa protein was immunoprecipitated with anti-UCS serum and shown to react with a monoclonal antibody against a conserved MSG epitope. To explore the functional role of the UCS in the trafficking of MSG, the nucleotide sequence encoding the UCS peptide was ligated to the 5' end of an MSG gene and incorporated into a recombinant baculovirus. Insect cells infected with the UCS-MSG hybrid gene expressed a 160-kDa protein which was N-glycosylated. By contrast, insect cells infected with a baculovirus carrying an MSG gene lacking the UCS expressed a nonglycosylated 130-kDa protein. These data suggest that in P. carinii, translation begins in the UCS to produce a pre-MSG protein, which is subsequently directed to the endoplasmic reticulum and processed to the mature form by proteolytic cleavage.

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^* Corresponding author. Mailing address: Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, 231 Bethesda Ave., ML 0524, Cincinnati, OH 45267-0524. Phone: (513) 558-0069. Fax: (513) 558-8474. E-mail: stringjr{at}uc.edu.

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