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Identification of a putative precursor to the major surface glycoprotein of Pneumocystis carinii
S M Sunkin1, M J Linke, F X McCormack
1Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524, USA.
Abstract:
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.
Insights
Translation of Pneumocystis carinii f. sp. carinii major surface glycoprotein (MSG) likely begins in the Upstream Conserved Sequence (UCS). This initiates a pre-MSG protein, crucial for its endoplasmic reticulum targeting and maturation.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Trafficking
Background:
- The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii is essential for the pathogen.
- MSG is encoded by a family of heterogeneous genes, with mRNAs sharing a common 5' Upstream Conserved Sequence (UCS).
Purpose of the Study:
- To investigate if translation of MSG mRNA initiates within the UCS.
- To elucidate the functional role of the UCS in MSG protein processing and trafficking.
Main Methods:
- Raised polyclonal antiserum against the UCS-encoded peptide.
- Used immunoprecipitation and Western blotting to detect P. carinii proteins.
- Employed recombinant baculovirus expression systems to study UCS function in insect cells.
Main Results:
- Anti-UCS serum recognized a 170 kDa P. carinii protein, distinct from mature 116 kDa MSG.
- Recombinant MSG lacking UCS produced a nonglycosylated 130 kDa protein in insect cells.
- Recombinant MSG with UCS produced an N-glycosylated 160 kDa protein, suggesting ER involvement.
Conclusions:
- Translation of MSG likely initiates in the UCS, forming a pre-MSG protein.
- The UCS plays a role in directing pre-MSG to the endoplasmic reticulum for post-translational modification and maturation.
- Proteolytic cleavage is implicated in generating the mature MSG from the precursor.