Targeting the glycome of major milk proteins
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The objectives of this thesis were to 1) explore the glycosylation of major bovine milk proteins, 2) investigate the effect of lactational stage on glycan structures and 3) examine how these documented changes may influence bioactivity. In order to achieve the first objective, we initially focused on the presence and activity of glycosidases in Irish bovine milk, which were monitored over the first three months of lactation (chapter 2). A low level of variation in enzyme activity between animals was noted and colostrum samples assayed showed the highest level of glycosidase activity. This decreased through transitional milk production to minimal but constant levels in mature milk. The most biologically relevant glycosidases in bovine milk as deteremined by this work are N-acetyl-β-D-glucosaminidase, α-L-fucosidase, α-galactosidase and N-acetyl-neuraminidase. The possibility that the elevated levels of enzymatic activity in colostrum may play a role in the digestion of bovine milk glycans in the infant mammal is discussed, focusing on their potential functionality as substitutes for bacterial glycosidases prior to colonisation by the gut microflora. In order to fulfil the second objective, bovine lactoferrin (bLF) was isolated from milk samples (3 cows, 13 timepoints over lactation) and glycosylation changes were profiled through the use of lectin microarrays and monosaccharide analysis (chapter 3). A large number of studies have to date focussed on the structure of the glycans associated with this glycoprotein particularly when isolated from mature milk, however there is limited information available on how these structures change over lactation. Substantial profile differences between early and late lactation are detailed in this chapter and overall, the data suggests that more diverse complex-type oligosaccharides structures are present on bLF during early lactation with an abundance of oligomannose type glycans in later lactation. The impact of the differences observed in the glycoprofiles of bLF from colostrum compared to mature milk is also discussed in this chapter. To achieve the third objective, the effect of this heterogeneous glycosylation pattern on bLF’s biological activity was first investigated. Chapter 4 employed SPR technology to compare the binding of bLF from colostrum (early lactation) and mature milk (late lactation) to a panel of pathogenic bacteria viii (Staphylococcus aureus, Escherichia coli, Cronobacter sakazakii, Streptococcus pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes and Salmonella typhimurium). LF has been shown in the literature to bind to various bacteria from both commensal and pathogenic communities. This bacterial binding activity has been linked to LF’s prebiotic and anti-infective activities in vivo. In Chapter 4, novel interactions were identified for C. sakazakii, S. pneumoniae and P. aeruginosa with the highest binding observed for mature milk bLF in all cases, with the exception of S. typhimurium. The difference in bacterial binding observed may be as a direct result of the varying glycosylation profiles. The abundance of mannose residues on mature milk bLF’s glycans may play a role as decoy receptors, mimicking the high-mannose type structures present on intestinal mucins and non-specifically binding pathogenic bacteria. Chapter 5 sought to expand on the third objective of the thesis and advance the knowledge on the role of bovine milk protein glycosylation in functionality of milk glycoproteins. Glycomacropeptide (GMP), a 64 amino acid peptide, derived from k-casein, was selected given the current interest in this glycopeptide as a prebiotic. GMP promoted the growth of Bifidobacterium longum subsp. infantis (B. infantis), a prototype infant strain which has been the focus of many prebiotic studies involving free milk oligosaccharides. This activity was lost following periodate treatment of the GMP (GMP-P), which disables biological recognition of the conjugated oligosaccharides. Transcriptional analysis of B. infantis following growth in media supplemented with 2mg/ml GMP revealed a substantial transcriptional response to GMP relative to bacteria cultured with 2mg/ml GMP-P supplementation of the media. Exposure to intact GMP resulted in a greater number of differentially expressed transcripts and larger fold changes versus the periodate treated test. These results suggest that the O-linked glycosylation of GMP is intrinsic to the growth stimulation of B. infantis by GMP. When the pool of differentially expressed transcripts displaying up-regulation following exposure to intact GMP were considered, two duplicated genomic islands containing glycoside hydrolase (family 25) genes and fibronectin type ІІІ binding domain proteins were identified. Homologues of this genomic arrangement were present in other Bifidobacterium species, which suggested that it may be a conserved domain for the utilisation of glycosylated peptides/proteins. This study provides insights into the molecular basis for the prebiotic effect of ix bovine milk GMP on B. infantis and highlights its potential as a functional food ingredient for the promotion of a beneficial gut microbiota. Overall, this research adds to our understanding of the structural and functional importance of milk protein glycosylation. It also highlights the potential of these compositionally complex molecules as ingredients which can be exploited by the food and pharmaceutical industry. Further research in this area, including characterisation of the genetic elements in the bovine mammary gland which influence protein glycosylation and expression is required to further our knowledge on the biological importance of milk glycoproteins.
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