Mouse cytomegalovirus (MCMV), a β-herpesvirus, encodes the m145 family of
glycoproteins. Several members of this family are predicted to adopt the MHC-I fold
although their amino acid sequences exhibit less than 30% identity to classical MHC-I
(MHC-Ia) proteins. Our aim was to determine how related the viral proteins are to MHCIa
and characterize them in terms of cellular expression, structure and function. We
studied the cellular localization of FLAG-tagged m17, M37, m145, m151, m152, m153
and m155 in transfected mouse fibroblasts. Flow cytometry analysis of transfected cells
showed that M37, m145, m151 and m153 localize predominantly to the cell surface,
whereas the majority of m17, m152 and m155 remain inside the cell. MHC-Ia proteins
require assembly with β2-microglobulin (β2m) and peptide for stable cell surface
expression. Transient transfection studies with β2m- or transporter associated with
antigen (TAP)-deficient cell lines revealed that M37, m145, m151 and m153 could be
expressed stably at the cell surface in the absence of β2m or TAP expression.
To generate protein material for crystallization screening we evaluated both bacterial and
insect cell expression systems. Although most m145 family members could be expressed in bacteria in insoluble inclusion bodies, none of the proteins could be accurately
refolded. Since M37, m151 and m153 are cell surface molecules with the potential to
bind receptors on host cells, we focused our structure determination efforts on them and
evaluated their expression in Drosophila S2 insect cells. The extracellular domains of all
three proteins expressed at significant levels, however, m151 tended to aggregate and precipitate over time. M37 and m153 were stable and could easily be purified to
homogeneity. Size exclusion chromatography and SDS-PAGE analysis of m153
suggested that it forms a non-covalent homodimer. Analytical ultracentrifugation
experiments confirmed this observation and provided an estimated molecular mass of
78.8 kDa. Enzymatic and mass spectrometry analyses showed that insect-expressed m153
is highly glycosylated. We tested a wide range of crystallization conditions for m153. It
formed very fragile crystals and after optimization we obtained several that diffracted to
2.3 Å. To determine the structure of m153, we prepared a seleno-methionine derivative in
insect cells, collected data on a single crystal and solved the phases by the single
anomalous dispersion method. The m153 model was refined at 2.4 Å resolution to final
Rcryst and Rfree of 23% and 27.9%, respectively.
The m153 homodimer is formed by two MHC-I-like heavy chains, each consisting of two
α-helices arranged on a platform of seven β-strands and an Ig-like α3 domain. The
monomers are arranged “head-to-tail”, with the α1α2 platform domain of one chain
interacting with the Ig-like α3 domain of the other. The α1 and α2 helices are closely juxtaposed and do not form a peptide binding groove. Three N-linked carbohydrate
residues were visualized in the crystal structure. Major deviations from the MHC-I fold
include an extended N-terminus, which originates next to the α3 domain, and an
elongated α2 helix (designated H2b) that reaches down towards the α3 domain. In
addition, m153 has two unique disulfide bonds, one between strands of the platform
domain and another that links the extended N-terminus to the H2b helix. Both unique
disulfide bonds were verified by mass spectrometry. The canonical Ig-fold disulfide bond is present in the α3 domain. Alanine mutation of four amino acids involved in interface
hydrogen bonds abolished m153 dimer formation, validating the dimer interface
visualized in the structure. The crystal structure of m153, together with the recently
reported m157 structure, confirms the MHC-I fold for the MCMV m145 family and
highlights shared structural features in the m145 family. We have demonstrated
dimerization of full-length m153 in mammalian cells by bimolecular fluorescence
complementation and co-immunoprecipitation studies. Further, we have shown that m153
is expressed at the surface of MCMV-infected cells at early times after infection.
To initiate a search for host ligands of m153, we generated a reporter cell line by
introducing an m153-human zeta chain fusion protein into 43.1 cells that contain an
NFAT-driven GFP construct. While a variety of mouse cell lines were unable to stimulate
the m153-reporter cells, coculture with mouse splenocytes specifically induced GFP
production in m153-reporters but not in the parental or control reporter cell lines. We
identified conventional CD11c+ and plasmacytoid dendritic cells (DCs) as the most
potent m153-reporter cell stimulating populations in the spleen. The stimulation was
shown to be m153-specific, dose- and cell contact-dependent. DCs derived from bonemarrow
cultures also potently stimulated the m153-reporter cells. Macrophages and NK
cells exhibited weaker stimulation of the reporter cells, indicating lower levels of ligand
or that only small subsets of the cells express a ligand. DCs from several mouse strains,
but not from the rat, stimulated m153-reporter cells. We evaluated DC surface phenotype
and migratory capacity after coculture with m153-reporter cells or on m153-coated
plates, but could not detect any changes induced specifically by the presence of m153.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/6768 |
| Date | 20 March 2009 |
| Creators | Mans, Janet |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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