Variability, Heritability and Environmental Determinants of Human Plasma N-Glycome Ana Knez ˇevic ´, † Ozren Polas ˇek, ‡ Olga Gornik, † Igor Rudan, §,| Harry Campbell, | Caroline Hayward, ⊥ Alan Wright, ⊥ Ivana Kolc ˇic ´, ‡ Niaobh O’Donoghue, # Jonathan Bones, # Pauline M. Rudd, # and Gordan Lauc* ,†,∇ Department of Biochemistry and Molecular Biology, University of Zagreb, Faculty of Pharmacy and Biochemistry, Zagreb, Croatia, Andrija S ˇ tampar School of Public Health, University of Zagreb Medical School, Zagreb, Croatia, Croatian Centre for Global Health, University of Split Medical School, Split, and Institute for Clinical Medical Research, University Hospital “Sestre Milosrdnice”, Zagreb, Croatia, Public Health Sciences, University of Edinburgh, Edinburgh EH8 9AG, United Kingdom, MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom, Dublin-Oxford Glycobiology Laboratory, NIBRT, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland, and Genos d.o.o., Trg Ljudevita Gaja 6, 31000 Osijek, Croatia Received September 8, 2008 Plasma glycans were analyzed in 1008 individuals to evaluate variability and heritability, as well as the main environmental determinants that affect glycan structures. By combining HPLC analysis of fluorescently labeled glycans with sialidase digestion, glycans were separated into 33 chromatographic peaks and quantified. A high level of variability was observed with the median ratio of minimal to maximal values of 6.17 and significant age- and gender-specific differences. Heritability estimates for individual glycans varied widely, ranging from very low to very high. Glycome-wide environmental determinants were also detected with statistically significant effects of different variables including diet, smoking and cholesterol levels. Keywords: Human plasma glycome • N-glycosylation • Glycan analysis • Variability of glycans • Heritability of glycans • Age-dependent changes Introduction Despite glycosylation being the most complex and abundant post-translational modification, we have only recently started to understand the importance of these complex oligosaccharide structures (glycans) attached to protein backbones. 1 This is not surprising since the branched structures of sugars make the analysis of glycoconjugates significantly more challenging than the analysis of linear DNA and protein sequences. However, a significant part of more than a half of all proteins, and of nearly all membrane and extracellular proteins, are glycans, 2 and to be able to understand the function of these proteins, we also have to understand their glycan moieties. Glycan synthesis is an extremely complex process that involves hundreds of different enzymes. 3 Some of these en- zymes are very specific and contribute to the synthesis of a limited number of structures on a small number of proteins, while others (like ER-glycosyltransferases involved in synthesis of N-glycans, or pathways that produce sugar nucleotides) affect thousands of different proteins. The intricate mechanisms by which the interplay of gene expression and intracellular localization of their products give rise to specific glycan structures is only starting to be understood. 4 N-glycosylation is essential for multicellular life and its complete absence is embryonically lethal. 5 Mutations that significantly reduce the potential to synthesize the N-glycan core result in a set of severe diseases named “congenital disorders of glycosylation”. 6 Mutations that result in the creation of a new glycosylation site can also be deleterious. A number of such mutations have been identified and it is predicted that up to 1.4% of all known disease-causing mis- sense mutations result in gains of glycosylation. 7,8 On the other hand, terminal variability in glycans is common (e.g., ABO blood groups) and contribute to the protein heterogeneity in a population that is advantageous for evading pathogens and adapting to changing environment. 9 Glycans display a much higher interspecies variability than proteins, 10–12 but glycome variability within a species has never been thoroughly examined. Structural and conformational aspects of glycans are very complex 13 and a small change in a glycan structure can have important functional consequences, indicating that variability in glycans might explain an important part of human phenotypic variability. The heritability of glycans * To whom correspondence should be addressed. Dr. Gordan Lauc, Department of Biochemistry and Molecular Biology, University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovac ˇic ´a 1, 10 000 Zagreb, Croatia. Fax, +385 1 639 4400; e-mail, glauc@pharma.hr. † University of Zagreb. ‡ University of Zagreb Medical School. § University of Split Medical School. | University of Edinburgh. ⊥ Western General Hospital. # University College Dublin. ∇ Genos d.o.o. 694 Journal of Proteome Research 2009, 8, 694–701 10.1021/pr800737u CCC: $40.75  2009 American Chemical Society Published on Web 11/26/2008