Immunology and Cell Biology (2008) 86, 631–632 & 2008 Australasian Society for Immunology Inc. All rights reserved 0818-9641/08 $32.00 www.nature.com/icb
CORRESPONDENCE
Response to Milland et al.: Carbohydrate residues downstream of the terminal Gala(1,3)Gal epitope modulate the specificity of xenoreactive antibodies Immunology and Cell Biology (2008) 86, 631–632; doi:10.1038/icb.2008.65; published online 9 September 2008
In their recently published paper, Milland et al.1 described a monoclonal antibody (15.101) that ‘binds cell surface expressed Gala(1,3)Gal produced by a1,3GT only weakly, whereas it binds intensely to Gala(1,3)Gal produced by iGb3S (that is, iGb3)’. This conclusion was based on flow cytometry staining of cell surface glycoconjugates expressed by human 293 cells transfected with either a1, 3GT or iGb3S, the latter of which uses only lactosylceramide (Galb1,4Glcb1,1Cer) as substrate. However, in an earlier study published by same group, Taylor et al.2 reported that the same iGb3S enzyme, in rat iGb3S-transfected CHO cells, produced a barely detectable amount of iGb3, as measured by thin layer chromatography analysis of glycosphingolipids (GSLs) extracted from those cells metabolically radiolabeled with 3H-Gal. In contrast, the authors reported the production of a large amount of poly-a-Gal-extended GSL structures, which were sensitive to coffee bean a-galactosidase digestion. In a separate study by another group, Keusch et al.3 studied rat iGb3S-transfected CHO cells by metabolically labeling them with 3H-GlcNAc, and reported the synthesis of an iGb5 structure, GalNAca1,3GalNAcb1,3Gala1,3 Galb1,4 Glcb1,1Cer, which was susceptible to digestion by chicken liver a-N-acetylgalactosaminidase. In addition, CHO cells transfected with rat iGb3S showed a strong binding to the monoclonal antibody, M1/22.21, which is specific for a terminal GalNAca1,3GalNAcb(Forssman GSL) sequence. We have reinvestigated the GSL expression by CHO–iGb3S cells using ESI-MSn (electrospray ionization ion trap mass spectrometry),4,5 and confirmed the clear presence of a series of poly-a-Gal-extended GSL struc-
tures (Figure 1). MSn analysis indicated that all of these poly a-Gal-extended GSLs contained a terminal Gala(1,3)Gal sequence (detailed results will be published elsewhere). We are currently examining other GSL components by ESI-MSn to identify GalNAca1, 3-terminated GSLs, such as iGb5. It is clear from these studies that biosynthesis of GSLs incorporating the iGb3 sequence is a far more complex process than that acknowledged by Milland et al.1 Thus, synthesis of iGb3 catalyzed by transfected rat iGb3S may be only a first step initiating the production of one or more series of GSLs with both Gala1,3Gala- and GalNAca1,3GalNAcb terminal sequences. It is not clear which GSLs are expressed by the iGb3S-transfected 293 cells Milland et al.1 have studied. The specificity of monoclonal antibody 15.101 has only been characterized against 6 types of complex carbohydrates (Gala1,3Gal-BSA, Gala1,3Galb1,4GlcNAcBSA, Gala1,3Galb1,4 Glc-BSA, Gala1,3Galb1, 4GlcNAcb1,3Galb1,4Glc-BSA, Galb1,4GlcNAc-BSA and Galb1,4Glc-BSA), which did not include those on important mammalian GSL structures, such as iGb5 and poly-a-Gal GSLs. Thus, we wish to report our reservations about the antibody specificity as claimed by Milland et al.1 that ‘the 15.101 mAb is a unique and promising reagent because of its preferential binding of the iGb3 glycolipid’, as the 15.101 antibody has been used to draw conclusions in several published papers.6–8 In addition, we wish to report that in certain types of cells, iGb3 may be converted to at least two series of GSL structures (iGb4, iGb5 and poly-Gal-GSLs), which are unlikely to be detected by an antiiGb3 antibody having the specificity claimed for 15.101.
In summary, we have identified at least five glycolipid structures in rat iGb3 enzymetransfected cells: iGb4, iGb5, Gala1,3Gala1, 3Galb1,4Glcb1,1Cer, Gala1,3Gala1,3Gala1, 3Galb1,4Glcb1,1Cer and Gala1,3Gala1,3Gala1, 3Gala1,3Galb1,4Glcb1,1Cer. In addition, Keusch et al.3 identified iGb5 structure in rat iGb3 enzyme-transfected cells. So there exist at least five different structures in rat iGb3 enzyme-transfected cells, which could potentially cross-react with 15.101 antibody, terminated by either Gal or GalNAc. As demonstrated by our data and a previous study from Sandrin and colleagues,2 iGb3 was almost completely converted to other glycolipids in rat iGb3 enzyme-transfected CHO cells. The majority of iGb3 has been converted to iGb4, iGb5, Gala1, 3Gala1,3Galb1,4Glcb1,1Cer, Gala1,3Gala1, 3Gala1,3Galb1,4Glcb1,1Cer, and Gala1,3Gala1,3Gala1,3Gala1,3Galb1,4Glcb1,1Cer, and other glycolipids have yet to be identified. As Sandrin and colleagues2 discussed in their earlier paper, the expression of these multiple GSLs is regulated by other glycosyltransferases present in CHO cells. The detailed expression and regulation of these GSLs in each specific cell type are extremely complex. As demonstrated by our data and a previous paper from Sandrin and colleagues,2 iGb3 is barely detectable in the cell transfects generated by both the Zhou and Sandrin and colleagues.2 We are capable of detecting iGb3 in rat iGb3 enzyme-transfected cells only by using sensitive, specific and generally applicable ion trap mass spectrometry technology (manuscript in preparation). Our results, as well as data from Sandrin and colleagues,2 have well demonstrated the fact that the iGb3 determinant can be rendered almost completely cryptic by further
Correspondence 632
Dapeng Zhou1 and Steven B Levery2 1Department
of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA and 2Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen N, Denmark E-mails:
[email protected] and
[email protected]
Figure 1 Poly-a-Gal GSLs expressed by CHO-iGb3S cells. CHO cells were stably transfected by a rat iGb3 synthase (CHO–iGb3S), and the neutral GSL fraction was purified as described.5 CHO cells transfected with a mock vector were used as negative control (CHO-mock). (a) Neutral GSLs from CHOiGb3S cells (lane 2) and CHO-mock cells (lane 3) were analyzed by thin layer chromatography separation followed by orcinol/sulfuric acid staining to visualize GSLs. Lane 1 was standard Gb3 (Matreya, Catalog number 1067, C60H103NO18, Pleasant Gap, PA, USA). (b) Neutral GSLs were permethylated as described.4,5 Electrospray ionization mass spectrometry was carried out in positive ion mode on a linear ion trap mass spectrometer (LTQ; ThermoFinnigan, San Jose, CA), using a nanoelectrospray source. MS1 molecular ions representing tetra-, penta- and hexaglycosylceramides were identified. MSn analysis indicated that their structures were (a) Gala1,3Gala1, 3Galb1,4Glcb1,1Cer (MS1 m/z 1531.2). (b): Gala1,3Gala1,3Gala1,3Galb1,4Glcb1,1Cer (MS1 m/z 1623.2, 1707.3, and 1735.4). (c) Gala1,3Gala1,3Gala1,3Gala1,3Galb1,4Glcb1,1Cer (MS1 m/z 1827.4, 1919.5 and 1939.5). Multiple molecular ion species represent heterogeneity with respect to fatty-N-acyl lipoforms. GSLs, glycosphingolipids.
glycosylation; thus the absence of cell-surface reactivity with even a well-validated 15.101 can never be used as the sole criterion for the lack of iGb3S expression. Without performing biochemical studies, it is incorrect to assume that transfection of iGb3 synthase will only result in iGb3 overexpression. In another recent paper,8 Sandrin and colleagues also referred to human cells transfected by rat iGb3 synthase as ‘human cells expressing iGb3’. Taylor et al.’s2 and our own biochemical studies indicate that these transfectants express only trace amount of iGb3, but high abundance of poly-a-Gal
Immunology and Cell Biology
GSLs, which are responsible for binding to anti-a-Gal IgG in human serum and complement-mediated lysis. ABBREVIATIONS a1,3GT, a-1,3 galactosyltransferase I; 3H-Gal, 3H-labeled galactose; 3H-GlcNAc, 3H-N-acetylglucosamine; GSL, glycosphingolipid; iGb3, isoglobotrihexosylceramide (Gala1,3Galb1, 4Glcb1,1Cer); iGb3S, iGb3 synthase; iGb5, isoForssman (GalNAca1,3GalNAcb1,3Gal a1,3Galb1,4Glcb1,1Cer); TLC, thin layer chromatography; ESI-MSn, electrospray ionization-ion trap mass spectrometry.
1 Milland J, Yuriev E, Xing PX, McKenzie IF, Ramsland PA, Sandrin MS. Carbohydrate residues downstream of the terminal Gala(1,3)Gal epitope modulate the specificity of xenoreactive antibodies. Immunol Cell Biol 2007; 85: 623–632. e-pub ahead of print 28 August 2007. 2 Taylor SG, McKenzie IF, Sandrin MS. Characterization of the rat a(1,3)galactosyltransferase: evidence for two independent genes encoding glycosyltransferases that synthesize Gala(1,3)Gal by two separate glycosylation pathways. Glycobiology 2003; 13: 327–337. e-pub ahead of print 17 December 2002. 3 Keusch JJ, Manzella SM, Nyame KA, Cummings RD, Baenziger JU. Expression cloning of a new member of the ABO blood group glycosyltransferases, iGb3 synthase, that directs the synthesis of isoglobo-glycosphingolipids. J Biol Chem 2000; 275: 25308–25314. 4 Li Y, Zhou D, Xia C, Wang PG, Levery SB. Sensitive quantitation of isoglobotriaosylceramide in the presence of isobaric components using electrospray ionization-ion trap mass spectrometry. Glycobiology 2008; 18: 166–176. e-pub ahead of print 28 November 2007. 5 Li Y, Teneberg S, Thapa P, Bendelac A, Levery SB, Zhou D. Sensitive detection of isoglobo and globo series tetraglycosylceramides in human thymus by ion trap mass spectrometry. Glycobiology 2008; 18: 158–165. e-pub ahead of print 3 December 2007. 6 Milland J, Christiansen D, Lazarus BD, Taylor SG, Xing PX, Sandrin MS. The molecular basis for Gala(1,3)Gal expression in animals with a deletion of the a1, 3galactosyltransferase gene. J Immunol 2006; 176: 2448–2454. 7 Milland J, Sandrin MS. ABO blood group and related antigens, natural antibodies and transplantation. Tissue Antigens 2006; 68: 459–466; review. 8 Christiansen D, Milland J, Mouhtouris E, Vaughan H, Pellicci DG, McConville MJ et al. Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT Cell development and transplantation. PLoS Biol 2008; 6: e172.
