Immunologic glycosphingolipidomics and NKT cell development in mouse thymus.
Invariant NKT cells are a hybrid cell type of Natural Killer cells and T cells, whose development is dependent on thymic positive selection mediated by double positive thymocytes through their recognition of natural ligands presented by CD1d, a nonpolymorphic, non-MHC, MHC-like antigen presenting molecule. Genetic evidence suggested that beta-glucosylceramide derived glycosphingolipids (GSLs) are natural ligands for NKT cells. N-butyldeoxygalactonojirimycin (NB-DGJ), a drug that specifically inhibits the glucosylceramide synthase, inhibits the endogenous ligands for NKT cells. Furthermore, we and others have found a beta-linked glycosphingolipid, isoglobotriaosylceramide (iGb3), is a stimulatory NKT ligand. The iGb3 synthase knockout mice have a normal NKT development and function, indicating that other ligands exist and remain to be identified. In this study, we have performed a glycosphingolipidomics study of mouse thymus, and studied mice mutants which are deficient in beta-hexosaminidase b or alpha-galactosidase A, two glycosidases that are up- and downstream agents of iGb3 turnover, respectively. Our mass spectrometry methods generated a first database for glycosphingolipids expressed in mouse thymus, which are specifically regulated by rate-limiting glycosidases. Among the identified thymic glycosphingolipids, only iGb3 is a stimulatory ligand for NKT cells, suggesting that large-scale fractionation, enrichment and characterization of minor species of glycosphingolipids are necessary for identifying additional ligands for NKT cells. Our results also provide early insights into cellular lipidomics studies, with a specific focus on the important immunological functions of glycosphingolipids.
Li
Yunsen
Y
Department of Melanoma Medical Oncology, Mass Spectrometry Core Facility, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77054, USA.
Thapa
Prakash
P
Hawke
David
D
Kondo
Yuji
Y
Furukawa
Keiko
K
Furukawa
Koichi
K
Hsu
Fong-Fu
FF
Adlercreutz
Dietlind
D
Weadge
Joel
J
Palcic
Monica M
MM
Wang
Peng G
PG
Levery
Steven B
SB
Zhou
Dapeng
D
D000818:Animals; D005700:Galactosyltransferases / Q000378:metabolism; D015870:Gene Expression; D023281:Genomics / Q000379:methods; D005915:Globosides / Q000276:immunology / Q000378:metabolism*; D006028:Glycosphingolipids / Q000276:immunology / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D051379:Mice; D018345:Mice, Knockout; D055611:Natural Killer T-Cells / Q000276:immunology* / Q000378:metabolism*; D013950:Thymus Gland / Q000166:cytology* / Q000276:immunology; D000519:alpha-Galactosidase / Q000235:genetics / Q000378:metabolism; D054821:beta-Hexosaminidase beta Chain / Q000235:genetics / Q000378:metabolism
D005915:Globosides; D006028:Glycosphingolipids; D008024:Ligands; C037097:isoglobotriaosylceramide; D005700:Galactosyltransferases; C413211:UDP-galactose beta-D-galactosyl-1,4-glycosylceramide alpha-1,3-galactosyltransferase; D000519:alpha-Galactosidase; D054821:beta-Hexosaminidase beta Chain