Immunoglobulin A N-glycosylation Presents Important Body Fluid-specific Variations in Lactating Mothers.

Secretory Immunoglobulin A (SIgA) is central to mucosal immunity: represents one of the main immunological mechanisms of defense against the potential attack of pathogens. During lactation SIgA is produced by plasmablasts in the mammary gland and is present in breast milk, playing a vital role in the passive immunity of the newborn. Interestingly, the different components of SIgA are highly N-glycosylated, and these N-Glycans have an essential role in health maintenance. In this work, we performed a glycomic study to compare N-glycosylation of SIgA purified from mature breast milk and saliva, and plasma IgA from the same lactating participants. Our results revealed a greater diversity than previously reported, with 89 glycan compositions that may correspond to over 250 structures. Among these glycans, 54 glycan compositions were characterized as body-fluid specific. Most of these unique N-Glycan compositions identified in SIgA from mature milk and IgA from plasma were fucosylated and both fucosylated and sialylated species, whereas in salivary SIgA the unique structures were mainly undecorated complex N-Glycans. In addition, we evaluated the effect of delivery mode on (S)IgA glycosylation. Lactating participants who had given birth by vaginal delivery presented an increased proportion of high mannose and fucosylated glycans in salivary SIgA, and selected high mannose, fucosylated, sialylated, and both fucosylated and sialylated glycans in plasma IgA, indicating that the hormonal changes during vaginal delivery could affect plasma and saliva IgA. These results reveal the structural details that provide a new dimension to the roles of (S)IgA N-Glycans in different tissues, and especially in maternal and new-born protection and infant development. The design of optimal recombinant IgA molecules specifically targeted to protect mucosal surfaces will need to include this dimension of structural detail.

Polymorphisms in genes in the SREBP1 signalling pathway and SCD are associated with milk fatty acid composition in Holstein cattle.

Genes in the sterol regulatory element-binding protein-1 (SREBP1) pathway play a central role in regulation of milk fat synthesis, especially the de-novo synthesis of saturated fatty acids. SCD, a SREBP-responsive gene, is the key enzyme in the synthesis of monounsaturated fatty acids in the mammary gland. In the present study, we discovered SNP in candidate genes associated with this signalling pathway and SCD to identify genetic markers that can be used for genetic and metabolically directed selection in cattle. We resequenced six candidate genes in the SREBP1 pathway (SREBP1, SCAP, INSIG1, INSIG2, MBTPS1, MBTPS2) and two genes for SCD (SCD1 and SCD5) and discovered 47 Tag SNP that were used in a marker-trait association study. Milk and blood samples were collected from Holstein cows in their 1st or 2nd parity at 100-150 days of lactation. Individual fatty acids from C4 to C20, saturated fatty acid (SFA) content, monounsaturated fatty acid content, polyunsaturated fatty acid content and desaturase indexes were measured and used to perform the asociation analysis. Polymorphisms in the SCD5 and INSIG2 genes were the most representative markers associated with SFA/unsaturated fatty acid (UFA) ratio in milk. The analysis of desaturation activity determined that markers in the SCD1 and SCD5 genes showed the most significant effects. DGAT1 K232A marker was included in the study to examine the effect of this marker on the variation of milk fatty acids in our Holstein population. The percentage of variance explained by DGAT1 in the analysis was only 6% of SFA/UFA ratio. Milk fat depression was observed in one of the dairy herds and in this particular dairy one SNP in the SREBP1 gene (rs41912290) accounted for 40% of the phenotypic variance. Our results provide detailed SNP information for key genes in the SREBP1 signalling pathway and SCD that can be used to change milk fat composition by marker-assisted breeding to meet consumer demands regarding human health, as well as furthering understanding of technological aspects of cows' milk.

Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures.

Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-Pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein, and bovine fetuin using nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC-Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan microheterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.