Glycosylation contributes to variability in expression of murine cytomegalovirus m157 and enhances stability of interaction with the NK-cell receptor Ly49H

Department of Pathology, Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242, USA.
European Journal of Immunology (Impact Factor: 4.03). 09/2010; 40(9):2618-31. DOI: 10.1002/eji.200940134
Source: PubMed


NK cell-mediated resistance to murine cytomegalovirus (MCMV) is controlled by allelic Ly49 receptors, including activating Ly49H (C57BL/6 strain) and inhibitory Ly49I (129 strain), which specifically recognize MCMV m157, a glycosylphosphatidylinositol-linked protein with homology to MHC class I. Although the Ly49 receptors retain significant homology to classic carbohydrate-binding lectins, the role of glycosylation in ligand binding is unclear. Herein, we show that m157 is expressed in multiple, differentially N-glycosylated isoforms in m157-transduced or MCMV-infected cells. We used site-directed mutagenesis to express single and combinatorial asparagine (N)-to-glutamine (Q) mutations at N178, N187, N213, and N267 in myeloid and fibroblast cell lines. Progressive loss of N-linked glycans led to a significant reduction of total cellular m157 abundance, although all variably glycosylated m157 isoforms were expressed at the cell surface and retained the capacity to activate Ly49H(B6) and Ly49I(129) reporter cells and Ly49H(+) NK cells. However, the complete lack of N-linked glycans on m157 destabilized the m157-Ly49H interaction and prevented physical transfer of m157 to Ly49H-expressing cells. Thus, glycosylation on m157 enhances expression and binding to Ly49H, factors that may impact the interaction between NK cells and MCMV in vivo where receptor-ligand interactions are more limiting.

Download full-text


Available from: Catherine A Forbes,
12 Reads
  • Source
    • "The retroviral transduction system including the pMX vectors, retroviral packaging cell line Platinum-E (PLAT-E), BWZ.36 cells expressing an inducible NFAT-lacZ reporter cassette and the derivative reporter line expressing Ly49H, Ly49HI129, or Ly49HIB6 have been previously described [16], [35]. The Ly49HI129 and Ly49HIB6 cell lines were a generous gift from Wayne Yokoyama (Washington University, St. Louis, MO, USA). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The murine cytomegalovirus-encoded protein m157 is a cognate ligand for both inhibitory and activating receptors expressed by natural killer cells. Additionally, m157 is expressed on the surface of infected cells by a glycophosphatidylinositol (GPI) anchor. Although endogenous GPI-anchored proteins are known to be ligands for the NK cell receptor, NKG2D, the contribution of the GPI anchor for viral m157 ligand function is unknown. To determine whether the GPI anchor for m157 is dispensable for m157 function, we generated m157 variants expressed as transmembrane fusion proteins and tested cells expressing transmembrane m157 for the capacity to activate cognate Ly49 receptors. We found that the GPI anchor is required for high-level cell surface expression of m157, and that the transmembrane m157 ligand retains the capacity to activate reporter cells and NK cells expressing Ly49H, as well as Ly49I(129) reporter cells, but with reduced potency. Importantly, target cells expressing the transmembrane form of m157 were killed less efficiently and failed to mediate Ly49H receptor downregulation on fresh NK cells compared to targets expressing GPI-anchored m157. Taken together, these results show that the GPI anchor for m157 facilitates robust cell surface expression, and that NK cells are sensitive to the altered cell surface expression of this potent viral evasin.
    PLoS ONE 06/2013; 8(6):e67295. DOI:10.1371/journal.pone.0067295 · 3.23 Impact Factor
  • Source
    • "Indeed, given the four putative N-glycosylation motifs (NXT/S) of m157, a variety of glycosylated isoforms were found to be expressed on the surface of MCMV-infected cells. Although these isoforms are also recognized by Ly49H, their binding stability and half-life are increased as compared to the unglycosylated m157 [43]. These results reveal a quite tolerant recognition of m157 by Ly49H where several amino acid substitutions are necessary in order to disrupt m157-Ly49H interactions. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Clinical and experimental data indicate that a subset of innate lymphocytes, natural killer (NK) cells, plays a crucial role in the response against herpesviruses, especially cytomegaloviruses (CMV). Indeed, in mice, NK cells, due to the expression of germline encoded Ly49 receptors, possess multiple mechanisms to recognize CMV infection. Classically, this results in NK cell activation and the destruction of the infected cells. More recently, however, this unique host-pathogen interaction has permitted the discovery of novel aspects of NK cell biology, implicating them in the regulation of adaptive immune responses as well as in the development of immunological memory. Here, we will concisely review the newly acquired evidence pertaining to NK cell Ly49-dependent recognition of MCMV-infected cell and the ensuing NK cell regulatory responses.
    BioMed Research International 05/2011; 2011(26):641702. DOI:10.1155/2011/641702 · 2.71 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The decisive events in the development of decidual cells (DC) are presented through examples of human and rodent decidua. Human decidua is formed by large decidual cells (LDC), endometrial granulated cells (eGC), and small decidual cells. The LDC form the main type of decidual membranes, which determine the morphological characteristics of the decidua as a tissue. Immediate precursor cells of LDC are located below the basement membrane of the uterine epithelium before and during implantation. At the next stage of differentiation, LDC acquire a spindle-like shape. Rodent LDC form an epithelium-like structure with gland properties at the terminal stage of differentiation. The single-cell structure of human decidua is a derivative of the epithelial organization of rodent decidua. Spindle-like rat LDC are characterized by a high level of protein, RNA, and DNA synthesis and by intensive proliferation. At the beginning of pregnancy, a cell proliferation predominates over cell loss. By Days 12-13 of rat pregnancy LDC loss reaches 80% per day. Terminally differentiated LDC (tLDC) disappear from decidua due to apoptosis. Apoptosis of tLDC and the exhaustion of their precursors account for the disappearance of LDC in the middle of rat pregnancy. Human term decidua is composed of living cells. Human LDC (hLDC) comprise the largest part of human decidual cells (hLDC). hLDC account for 60-90% of hDC but their relative amount can decrease to 35% in the case of significant cell loss under unfavorable conditions. A decrease of LDC is not accompanied by DC proliferation. The lack of ability of decidua to compensate for DC loss suggests DC is a growing type of cell population without cambial cells. LDC function largely by blebbing and budding. Human and rat endometrial granulated cells (eGC) are characterized by a low level of natural killer (NK) activity and a high level of natural suppressor (NS) activity. The combination of NK and NS properties is characteristic of the eGC immunoregulatory function. Other functions of decidua include control of inflammation and trophoblast growth and expansion in the uterus. The life span of decidual cells is limited by pregnancy.
    International Review of Cytology 02/2003; 227:1-63. DOI:10.1016/S0074-7696(03)01007-6 · 9.00 Impact Factor
Show more