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37-OR: Complexity of the ligands presented by human CD1d
Abstracts
S117
37-OR
COMPLEXITY OF THE LIGANDS PRESENTED BY HUMAN CD1D. Daryl Cox,1 Lisa Fox,2 Runying Tian,1 Wilfried Bardet,1 Matthew Skaley,1 Danijela Mojsilovic,1 Jenny Gumperz,2 William Hildebrand.1 1University of Oklahoma HSC; 2University of Wisconsin. Aim: CD1 molecules are MHC Class I-like glycoproteins that present lipids instead of peptides at the cell surface. The breadth of the endogenously loaded lipids displayed by CD1 molecules has not been reported. Therefore, our primary aim is to utilize our system of soluble HLA production to prepare milligram quantities of sCD1d from which the breadth of the ligands presented by human CD1d can be determined. Methods: Utilizing our method of producing soluble MHC Class I molecules, we produced milligram quantities of soluble human CD1d. Herein we report the isolation of soluble human CD1d loaded with endogenous lipids using affinity chromatography. The lipid cargo of the CD1d is removed via a Bligh and Dyer extraction. Subsequently, the extracted lipids are subjected to normal-phase HPLC which separates the lipids by exploiting the differences in their polar head moieties. Mass spectroscopic analysis of each fraction is used to determine the structure of the CD1d ligands. Results: Mass spectroscopic analysis of the HPLC fractions revealed the presence of a wide variety of lipids, which fall into two basic classes— glycerolipids and sphingolipids. Within the glycerolipid category, CD1d displayed: both the monoacyl and diacyl glycerolipids and cardiolipins. Whereas, within the sphingolipid group CD1d exhibited sphingomyelins and sialic acid containing glycosphingolipids. Conclusions: The complexity of endogenous CD1d ligands have been explored by successfully producing milligram quantities of sCD1d, from which the endogenous ligands were extracted, separated by normal phase HPLC and subsequently characterized by mass spectroscopic analysis. The lipids characterized are of the glycerolipid and sphingolipid categories.
38-OR
PINPOINTING A SELECTIVE SWEEP TO THE CHIMPANZEE MHC CLASS I REGION BY COMPARATIVE GENOMICS. Natasja G. de Groot,1 Corrine M.C. Heijmans,1 Nanine de Groot,1 Edmond J. Remarque,1 Maxime Bonhomme,2 Brigitte Crouau-Roy,2 Ronald E. Bontrop.1 1CG&R, BPRC, Rijswijk, Netherlands; 2EDB, Universite Paul Sabatier, Toulouse, France. Aim: Together with humans, chimpanzees are the only species that can be infected with human immunodeficiency virus type 1 (HIV-1), but chimpanzees seem to be relative resistant to the development of acquired immuno deficiency syndrome (AIDS). Furthermore it is known that chimpanzees produce effective CTL responses to conserved HIV-1 epitopes. These observations led us put forward the hypothesis that the selective sweep targeting the chimpanzee Mhc class I region was presumably caused by HIV-1/SIVcpz or a related ancestral retrovirus. With long-scale genomic analysis it was aimed to investigate the extent of the repertoire reduction. Methods: Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees, supplemented with multilocus demographic analyses. Results: Chimpanzees are as a species much older than humans and as such had more time to generate variation. The analyzed microsatellite markers mapping outside the Mhc region reflect this. Whereas for almost all analyzed microsatellite markers located inside the Mhc region humans show much more variation than chimpanzees. Multilocus demographic analyses underscore that the chimpanzee Mhc class I region is targeted by the selective sweep. Conclusions: The microsatellite analyses together with the multilocus demographic analyses pinpoints the selective sweep to the chimpanzee Mhc class I region, with a strong repertoire reduction mapping in the vicinity of the Mhc-B locus. Although, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep, it had not sufficient time to erase the signal of the sweep.
S117
37-OR
COMPLEXITY OF THE LIGANDS PRESENTED BY HUMAN CD1D. Daryl Cox,1 Lisa Fox,2 Runying Tian,1 Wilfried Bardet,1 Matthew Skaley,1 Danijela Mojsilovic,1 Jenny Gumperz,2 William Hildebrand.1 1University of Oklahoma HSC; 2University of Wisconsin. Aim: CD1 molecules are MHC Class I-like glycoproteins that present lipids instead of peptides at the cell surface. The breadth of the endogenously loaded lipids displayed by CD1 molecules has not been reported. Therefore, our primary aim is to utilize our system of soluble HLA production to prepare milligram quantities of sCD1d from which the breadth of the ligands presented by human CD1d can be determined. Methods: Utilizing our method of producing soluble MHC Class I molecules, we produced milligram quantities of soluble human CD1d. Herein we report the isolation of soluble human CD1d loaded with endogenous lipids using affinity chromatography. The lipid cargo of the CD1d is removed via a Bligh and Dyer extraction. Subsequently, the extracted lipids are subjected to normal-phase HPLC which separates the lipids by exploiting the differences in their polar head moieties. Mass spectroscopic analysis of each fraction is used to determine the structure of the CD1d ligands. Results: Mass spectroscopic analysis of the HPLC fractions revealed the presence of a wide variety of lipids, which fall into two basic classes— glycerolipids and sphingolipids. Within the glycerolipid category, CD1d displayed: both the monoacyl and diacyl glycerolipids and cardiolipins. Whereas, within the sphingolipid group CD1d exhibited sphingomyelins and sialic acid containing glycosphingolipids. Conclusions: The complexity of endogenous CD1d ligands have been explored by successfully producing milligram quantities of sCD1d, from which the endogenous ligands were extracted, separated by normal phase HPLC and subsequently characterized by mass spectroscopic analysis. The lipids characterized are of the glycerolipid and sphingolipid categories.
38-OR
PINPOINTING A SELECTIVE SWEEP TO THE CHIMPANZEE MHC CLASS I REGION BY COMPARATIVE GENOMICS. Natasja G. de Groot,1 Corrine M.C. Heijmans,1 Nanine de Groot,1 Edmond J. Remarque,1 Maxime Bonhomme,2 Brigitte Crouau-Roy,2 Ronald E. Bontrop.1 1CG&R, BPRC, Rijswijk, Netherlands; 2EDB, Universite Paul Sabatier, Toulouse, France. Aim: Together with humans, chimpanzees are the only species that can be infected with human immunodeficiency virus type 1 (HIV-1), but chimpanzees seem to be relative resistant to the development of acquired immuno deficiency syndrome (AIDS). Furthermore it is known that chimpanzees produce effective CTL responses to conserved HIV-1 epitopes. These observations led us put forward the hypothesis that the selective sweep targeting the chimpanzee Mhc class I region was presumably caused by HIV-1/SIVcpz or a related ancestral retrovirus. With long-scale genomic analysis it was aimed to investigate the extent of the repertoire reduction. Methods: Comparison of genetic variation at microsatellite markers mapping inside and outside the Mhc region was carried out in humans and chimpanzees, supplemented with multilocus demographic analyses. Results: Chimpanzees are as a species much older than humans and as such had more time to generate variation. The analyzed microsatellite markers mapping outside the Mhc region reflect this. Whereas for almost all analyzed microsatellite markers located inside the Mhc region humans show much more variation than chimpanzees. Multilocus demographic analyses underscore that the chimpanzee Mhc class I region is targeted by the selective sweep. Conclusions: The microsatellite analyses together with the multilocus demographic analyses pinpoints the selective sweep to the chimpanzee Mhc class I region, with a strong repertoire reduction mapping in the vicinity of the Mhc-B locus. Although, naturally occurring recombination events allowed the establishment of haplotype diversity after the sweep, it had not sufficient time to erase the signal of the sweep.