Chemical characterisation of the oligosaccharides in hooded seal (Cystophora cristata) and Australian fur seal (Arctocephalus pusillus doriferus) milk

Carbohydrates were extracted from hooded seal milk, Crystophora cristata (family Phocidae). Free oligosaccharides were separated by gel filtration and then purified by ion exchange chromatography, gel filtration and preparative thin layer or paper chromatography and their structures determined by ¹H-NMR. The hooded seal milk was found to contain inositol and at least nine oligosaccharides, most of which had lacto-N-neotetraose or lacto-N-neohexaose as core units, similar to those in milk of other species of Carnivora such as bears (Ursidae). Their structures were as follows: Gal(β1-4)Glc (lactose); Fuc(α1-2)Gal(β1-4)Glc (2′-fucosyllactose); Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (lacto-N-neotetraose); Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (lacto-N-fucopentaose IV); Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(1-4)Glc (lacto-N-neohexaose); Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monofucosyl lacto-N-neohexaose a); Gal(β1-4)GlcNAc(β1-3)[Fuc(α1-2)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (monofucosyl lacto-N-neohexaose b); Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)[Fuc(α1-2)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (difucosyl lacto-N-neohexaose); Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (para lacto-N-neohexaose); Fuc(α1-2)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)GlcNAc(β1-3)Gal(β1-4)Glc (monofucosyl para lacto-N-neohexaose). Milk of the Australian fur seal, Arctophalus pusillus doriferus (family Otariidae) contained inositol but no lactose or free oligosaccharides. These results, therefore, support the hypothesis that the milk of otariids, unlike that of phocids, contains no free reducing saccharides.

Bovine Muc1 is a highly polymorphic gene encoding an extensively glycosylated mucin that binds bacteria

The bovine Muc1 protein is synthesized by mammary epithelial cells and shed into milk as an integral component of the milk fat globule membrane; however, the structure and functions of this mucin, particularly in relation to lactation, are poorly defined. The objectives of this investigation were to investigate the Muc1 gene and protein structures in the context of lactation and to test the hypothesis that Muc1 has a role in innate immune defense. Polymerase chain reaction analysis of genomic DNA from 630 cattle revealed extensive polymorphism in the variable number of tandem repeats (VNTR) in the bovine Muc1 gene. Nine allelic
variants spanning 7 to 23 VNTR units, each encoding 20 AA, were identified. Three alleles, containing 11, 14, and 16 VNTR units, respectively, were predominant. In addition, a polymorphism in one of the VNTR units has the potential to introduce a unique site for N-linked glycosylation. Statistical analysis indicated weak associations between the VNTR alleles and milk protein and fat percentages in a progeny-tested population of Holstein-Friesian dairy cattle. No association with somatic cell count could be demonstrated. Bovine Muc1 was purified from milk fat globule membranes and characterized. The protein was highly glycosylated, primarily with O-linked sialylated T-antigen [Neu5Ac(α2–3)-Gal(β1–3)-GalNAcα1] and, to a lesser extent, with N-linked oligosaccharides, which together accounted for approximately 60% of the apparent mass of Muc1. Purified bovine Muc1 directly bound fluorescently labeled Escherichia coli BioParticles (Invitrogen, Mount Waverley, Australia) and inhibited their binding to bovine mammary epithelial cells grown in vitro.

Conservation of the ST6Gal I gene and its expression in the mammary gland

Milk sialoglycoconjugates can protect the gastrointestinal tract of the suckling neonate by competitively binding to invading pathogens and promoting growth of beneficial flora, and their potential role in postnatal brain development is of particular interest in human infant nutrition. Although the concentration and the distribution of sialoglycoconjugates have been extensively studied in the milk of various species, the investigation of sialyltransferase gene expression in the mammary gland, in the context of lactation, has been limited. The sialyltransferase enzyme ST6Gal I transfers sialic acid from CMP-sialic acid to type 2 (Galβ1,4GlcNAc) free disaccharides or the termini of N- or O-linked oligosaccharides using an α2,6-linkage. Expression of the ST6Gal I gene is primarily regulated at the level of transcription through the use of several cell and development- specific promoters, producing transcripts with divergent 5′ untranslated regions (UTR). In the mouse mammary gland, the novel 5′UTR exon (L) appears to be associated with a drastic increase in ST6Gal I gene expression during lactation. We find that rats also possess an exon (L), suggesting conservation of this regulatory mechanism in rodents. In contrast, an exon (L)-containing transcript was not detected in the lactating bovine or human mammary gland. We also observed a trend of increasing ST6Gal I gene expression in the bovine mammary gland, culminating in involution. This is in contrast to species such as mice where the greatest change in ST6Gal I gene expression occurs between pregnancy and lactation, suggesting different roles in rodents vs. other mammals for α2,6-sialylated oligosaccharides present in milk.

Structural characterisation and bioactivities of hybrid carrageenan-like sulphated galactan from red alga Furcellaria lumbricalis

The red alga, Furcellaria lumbricalis from the coast of the Prince Edward Island (PEI) in Atlantic Canada, was extracted with hot water and fractionated with 0.125. M KCl to obtain a carrageenan-like polysaccharide. The polysaccharide was further purified on ion-exchange and gel-permeation chromatography to yield a fraction (FB1) of uniform size and charge, with an average molecular weight of 428. kD. Oligosaccharides generated with acid hydrolysis of FB1 were sequenced using the electrospray ionisation collision induced dissociation tandem mass spectrometry (ES-CID-MS/MS) technique. On the basis of chemical and spectroscopic analysis, FB1 was characterised to be composed of 1,4-linked 3,6-anhydro-galactose (40%), 1,3-linked 4-sulphated-galactose (30%), 1,3-linked galactose (20%), 1,4-linked galactose (8%) and 1,4-linked 3-O-methyl-galactose (2%), which makes it be a novel sulphated galactan hybrid. The β-secretase (BACE) inhibition and immunomodulation activities of FB1-derived oligosaccharides were evaluated in vitro.

Identification of unprecedented anticancer properties of high molecular weight biomacromolecular complex containing bovine lactoferrin (HMW-bLf)

With the successful clinical trials, multifunctional glycoprotein bovine lactoferrin is gaining attention as a safe nutraceutical and biologic drug targeting cancer, chronic-inflammatory, viral and microbial diseases. Interestingly, recent findings that human lactoferrin oligomerizes under simulated physiological conditions signify the possible role of oligomerization in the multifunctional activities of lactoferrin molecule during infections and in disease targeting signaling pathways. Here we report the purification and physicochemical characterization of high molecular weight biomacromolecular complex containing bovine lactoferrin (≥250 kDa), from bovine colostrum, a naturally enriched source of lactoferrin. It showed structural similarities to native monomeric iron free (Apo) lactoferrin (∼78-80 kDa), retained anti-bovine lactoferrin antibody specific binding and displayed potential receptor binding properties when tested for cellular internalization. It further displayed higher thermal stability and better resistance to gut enzyme digestion than native bLf monomer. High molecular weight bovine lactoferrin was functionally bioactive and inhibited significantly the cell proliferation (p<0.01) of human breast and colon carcinoma derived cells. It induced significantly higher cancer cell death (apoptosis) and cytotoxicity in a dose-dependent manner in cancer cells than the normal intestinal cells. Upon cellular internalization, it led to the up-regulation of caspase-3 expression and degradation of actin. In order to identify the cutting edge future potential of this bio-macromolecule in medicine over the monomer, its in-depth structural and functional properties need to be investigated further.