Gillian E. Norris

In this review, we have collected and analysed collagen glycosylation data in an effort to better understand its possible function in fibrillogenesis. Towards that end, we have identified glycosylation sites in many different fibril-forming collagen molecules (Types I, II, III, V, XI and XXIV) and, more importantly, the glycan itself as well as the percentage of collagen molecules in the sample that contain it. As the numbers of quantitative analyses of different collagen types from different species and tissues taken under different physiological conditions grows, a more accurate picture is emerging of the role that collagen O-glycosylation may play in fibrillogenesis. It is becoming increasingly clear that glycosylation does play an important part in the initial stages of the axial assembly of collagen molecules as well as in the lateral control of fibril size. Much remains unknown of the precise mechanisms involved, however, and further detailed analyses will be required before a complete picture of the various in vivo roles played by the glycans emerges.

Glycocin F (GccF), a ribosomally synthesized, post-translationally modified peptide secreted by Lactobacillus plantarum KW30, rapidly inhibits the growth of susceptible bacteria at nanomolar concentrations. Previous studies have highlighted structural features important for its activity and have shown the absolute requirement for the Ser18 O-linked GlcNAc on the eight-residue loop linking the two short helices of the (C-X6-C)(2) structure. Here, we show that an ostensibly very small chemical modification to Ser18, the substitution of the C-alpha proton with a methyl group, reduces the antimicrobial activity of GccF 1000-fold (IC50 1.5 mu M cf. 1.5 nM). A comparison of the GccF(alpha-methylSer18) NMR structure (PDB 8DFZ) with that of the native protein (PDB 2KUY) showed a marked difference in the orientation and mobility of the loop, as well as a markedly different positioning of the GlcNAc, suggesting that loop conformation, dynamics, and glycan presentation play an important role in the interaction of GccF with as yet unknown but essential physiological target molecules.

The entire 7-gene cluster for the diglycosylated bacteriocin glycocin F (GccF), including the natural promoters responsible forgccgene expression, has been ligated into theEscherichia coli-lactic acid bacteria (LAB) shuttle vector pRV610 to produce the easily modifiable 11.2-kbp plasmid pRV610gccfor the efficient production of glycocin F analogues. In contrast to the refactoring approach, chemical synthesis, or chemoenzymatic synthesis, all of which have been successfully used to probe glycocin structure and function, this plasmid can also be used to probein vivothe evolutionary constraints on glycocin scaffolds and their processing by the maturation pathway machinery, thus increasing understanding of the enzymes involved, the order in which they act, and how they are regulated.

Yeast impact homolog 1 (Yih1), or IMPACT in mammals, is part of a conserved regulatory module controlling the activity of General Control Nonderepressible 2 (Gcn2), a protein kinase that regulates protein synthesis. Yih1/IMPACT is implicated not only in many essential cellular processes, such as neuronal development, immune system regulation and the cell cycle, but also in cancer. Gcn2 must bind to Gcn1 in order to impair the initiation of protein translation. Yih1 hinders this key Gcn1‐Gcn2 interaction by binding to Gcn1, thus preventing Gcn2‐mediated inhibition of protein synthesis. Here, we solved the structures of the two domains of Saccharomyces cerevisiae Yih1 separately using Nuclear Magnetic Resonance and determined the relative positions of the two domains using a range of biophysical methods. Our findings support a compact structural model of Yih1 in which the residues required for Gcn1 binding are buried in the interface. This model strongly implies that Yih1 undergoes a large conformational rearrangement from a latent closed state to a primed open state to bind Gcn1. Our study provides structural insight into the interactions of Yih1 with partner molecules.

Cerebrosides (Crb; including glucosylceramide and galactosylceramide) and lactosylceramide (LacCer) are structurally complex lipids found in many eukaryotic cell membranes, where they play important roles in cell growth, apoptosis, cell recognition and signaling. They are also found in mammalian milk as part of the milk fat globule membrane (MFGM), making milk an important dietary component for the rapidly growing infant. This study reports the development of a robust analytical method for the identification and characterization of 44 Crb and 23 LacCer molecular species in milk, using high performance liquid chromatography–tandem mass spectrometry in data-dependent acquisition mode. For the first time, it also compares the distributions of these species in human and bovine milks, a commercial MFGM-enriched dairy ingredient (MFGM Lipid 100) and commercial standards purified from bovine milk. A method for quantifying Crb and LacCer in milk using mass spectrometry in neutral loss scan mode was developed and validated for human milk, bovine milk and MFGM Lipid 100. Human milk was found to contain approximately 9.9–17.4 µg Crb/mL and 1.3–3.0 µg LacCer/mL, whereas bovine milk (pooled milk from a Friesian herd) contained 9.8–12.0 and 14.3–16.2 µg/mL of these lipids, respectively. The process used to produce MFGM Lipid 100 was shown to have enriched these components to 448 and 1036 µg/g, respectively. No significant changes in the concentrations of both Crb and LacCer were observed during lactation.

In order to protect their environmental niche, most bacteria secret antimicrobial substances designed to target specific bacterial strains that are often closely related to the producer strain. Bacteriocins, small, ribosomally synthesised antimicrobial peptides, comprise a class of such substances and can either inhibit (bacteriostatic) or kill (bactericidal) target cells. Glycocins are a class of bacteriocin that are post-translationally modified by one or more carbohydrate moieties that are either β-O-linked to either a serine or threonine and/or β-S-linked to a cysteine. The solution nuclear magnetic resonance structure (NMR) of the glycocin ASM1 (produced by Lactobacillus plantarum A-1), an orthologue of GccF, has been determined. In both structures, the disulfide bonds are essential for activity and restrict the mobility of the N-acetyl-glucosamine (GlcNAc) attached to Ser-18 (O-linked), compared to the much more flexible GlcNAc moiety on Cys-43 (S-linked). Interestingly, despite 88% sequence identity, the helical structure of ASM1 is less pronounced which appears to be consistent with the far ultra-violet circular dichroism (UV CD) spectra.

Here, we report on the biochemical characterisation of a new glycosylated bacteriocin (glycocin), ASM1, produced by Lactobacillus plantarum A‐1 and analysis of the A‐1 bacteriocinogenic genes. ASM1 is 43‐amino acids in length with Ser18‐O‐ and Cys43‐S‐linked N‐acetylglucosamine moieties that are essential for its inhibitory activity. Its only close homologue, glycocin F (GccF), has five amino acid substitutions all residing in the flexible C‐terminal ‘tail’ and a lower IC₅₀ (0.9 nM) compared to that of ASM1 (1.5 nM). Asm/gcc genes share the same organisation (asmH←→asmABCDE→F), and the asm genes reside on an 11,905‐bp plasmid dedicated to ASM1 production. The A‐1 genome also harbours a gene encoding a ‘rare’ bactofencin‐type bacteriocin. As more examples of prokaryote S‐GlcNAcylation are discovered, the functions of this modification may be understood.