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Item The effect of microwave-assisted heating on bioactive and immunological compounds in donor human milk(Elsevier Ltd, 2022-05-01) Leite JAS; Robinson RC; Salcedo J; Ract JNR; Quintal VS; Tadini CC; Barile DLow-Temperature Long-Time pasteurization (LTLT) is normally applied in donor human milk from Human Milk Banks (HMBs) to guarantee microbiological safety; however, this treatment can modify the protein structures, decreasing their beneficial effects. Thus, this study aimed to determine the impact of microwave-assisted heating on the concentration of key biological compounds in donor human milk to verify whether a microwave heating technique can be used as an alternative to LTLT pasteurization in Human Milk Banks. The concentrations of oligosaccharides, immunoglobulins, lactoferrin and fatty acids were monitored in raw donor milk and after processing to assess the impact of the microwave and LTLT treatments. The concentration of oligosaccharides was determined by HPAEC-PAD, immunoglobulins and lactoferrin were quantified using ELISA kits and fatty acids were quantified by gas chromatography. Oligosaccharides and fatty acids were not significantly affected (p > 0.05) by LTLT and microwave processes; however, immunoglobulins and lactoferrin concentrations were better preserved when microwave-assisted heating was applied. For this reason, microwave-assisted heating can be considered a promising alternative to LTLT pasteurization of donor human milk in Human Milk Banks.Item A common regulatory haplotype doubles lactoferrin concentration in milk.(BioMed Central Ltd, 2024-03-28) Lopdell TJ; Trevarton AJ; Moody J; Prowse-Wilkins C; Knowles S; Tiplady K; Chamberlain AJ; Goddard ME; Spelman RJ; Lehnert K; Snell RG; Davis SR; Littlejohn MDBACKGROUND: Bovine lactoferrin (Lf) is an iron absorbing whey protein with antibacterial, antiviral, and antifungal activity. Lactoferrin is economically valuable and has an extremely variable concentration in milk, partly driven by environmental influences such as milking frequency, involution, or mastitis. A significant genetic influence has also been previously observed to regulate lactoferrin content in milk. Here, we conducted genetic mapping of lactoferrin protein concentration in conjunction with RNA-seq, ChIP-seq, and ATAC-seq data to pinpoint candidate causative variants that regulate lactoferrin concentrations in milk. RESULTS: We identified a highly-significant lactoferrin protein quantitative trait locus (pQTL), as well as a cis lactotransferrin (LTF) expression QTL (cis-eQTL) mapping to the LTF locus. Using ChIP-seq and ATAC-seq datasets representing lactating mammary tissue samples, we also report a number of regions where the openness of chromatin is under genetic influence. Several of these also show highly significant QTL with genetic signatures similar to those highlighted through pQTL and eQTL analysis. By performing correlation analysis between these QTL, we revealed an ATAC-seq peak in the putative promotor region of LTF, that highlights a set of 115 high-frequency variants that are potentially responsible for these effects. One of the 115 variants (rs110000337), which maps within the ATAC-seq peak, was predicted to alter binding sites of transcription factors known to be involved in lactation-related pathways. CONCLUSIONS: Here, we report a regulatory haplotype of 115 variants with conspicuously large impacts on milk lactoferrin concentration. These findings could enable the selection of animals for high-producing specialist herds.Item A mutational analysis of the hinge region of the N-lobe of lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1996) Shewry, Steven ChristopherLactoferrin is an 80kDa iron binding glycoprotein that is found as a major component of human milk, as well as in many other exocrine solutions. Lactoferrin binds reversibly, and with high affinity, 2 Fe3+ ions with 2 synergistic C032- ions. The crystal structure shows that the polypeptide chain is folded into two similar lobes, each binding one Fe3+ and CO32- ion. In the metal free state, the N-terminal lobe has been found to adopt an open structure, with rotation occurring around two residues found in separate beta strands located at the back of the binding site in a region referred to as the hinge. A sequence alignment of these two strands over the greater transferrin family shows a very high level of conservation particularly of the two residues at the centre of this rotation (Pro 251 and Thr 90). The N-terminal half of human lactoferrin (LfN) has been constructed, expressed and the crystal structure determined. In an attempt to understand the importance of the conservation of these two residues, and their effect on binding, a mutational analysis was initiated. Oligonucleotide site-directed mutagenesis has been used to construct mutants in the cDNA encoding for human lactoferrin using the M13 bacteriophage. The mutant cDNA was transformed into a mammalian expression vector (pNUT). After transfection of the pNUT vector into baby hamster kidney cells (BHK), the mutant proteins were expressed and purified from the culture medium using a CM-sephadex ion-exchange column. Absorption maxima and pH-dependent iron-release experiments were carried out on the mutants. The data shows that the mutants behave essentially the same as LfN, the exception being P251G which appears to release the iron over a shorter pH range. The reason for this is not yet fully understood. The crystal structure of P251A in the iron-bound form was solved by molecular replacement using the structure of LfN as the starting model. The structure of P251A was refined using data between 20.0 and 2.0 Ǻ. The current model has good geometry and has an R-factor of 18.6 %. Analysis of the structure shows that it is essentially identical to that of the LfN structure. Although the structure of the iron-free form has not been determined, it appears that changes to the hinge region of the N-lobe of lactoferrin do not affect the iron-binding or structural characteristics of the protein.Item Crystallographic and physicochemical studies on anion-binding and deglycosylation of human lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy in Chemistry at Massey University(Massey University, 1995) Baker, Heather MaryLactoferrin, a monomeric 80 kDa glycoprotein, is a major component of human milk and is found in many other exocrine secretions as well as in the neutrophilic granules of white blood cells. A member of the transferrin family of iron-binding proteins, lactoferrin has the ability to bind tightly but reversibly 2Fe3+ ions with the concomitant binding of 2CO32- ions. Crystal structure studies clearly demonstrate that the polypeptide chain is folded into two similar lobes, representing the N- and the C-terminal halves of the protein, and that each lobe contains one of the two very similar iron-binding sites. Transferrins also show considerable versatility in their binding properties, being able to bind many metal ions in place of Fe3+ and anions in place of CO32-. Differences between the two sites become more pronounced, however, with the substitution of non-native metals and anions, and the origins of this inequivalence have long been debated. To investigate the means by which lactoferrin can accommodate anions larger than carbonate, the diferricdioxalatolactoferrin (Fe2(C2O4)2Lf) complex was prepared and crystallised. The crystals, which were isomorphous with those of Fe2(CO3)2Lf, were used to collect a complete 2.4 Ǻ data set at the Photon Factory (Japan) synchrotron source. The structure was refined by restrained least squares methods to a final R factor of 0.196 for all 31758 reflections in the resolution range 8.0 to 2.4 Ǻ. The polypeptide folding and domain closure were identical to those of the native Fe2(CO3)2Lf. In contrast to the carbonate complex, however, in which the two binding sites appear almost identical, with the carbonate coordinating in a symmetrical bidentate mode to each iron, when oxalate is the anion, the coordination around the metal differs between the N- and the C-lobe. In the C-lobe, the oxalate has a symmetrical 1,2-bidentate coordination to the iron, but in the N-lobe this coordination is quite asymmetric (O1ox-Fe = 1.87 Ǻ, O2ox-Fe = 2.55 Ǻ). Analysis of the structure indicates that the stereochemistry of the oxalate coordination to the iron is influenced by the position of the anion-binding arginine. The position this arginine can adopt in each lobe is, in turn, influenced by residues more remote from the iron site and which differ between the N- and C-lobes. All lactoferrins so far characterised are glycoproteins, but the importance of the glycan chains for structure and/or function has yet to be established. Enzymatic methods were used to deglycosylate human and bovine lactoferrins, and the native deglycosylated forms of the human protein were compared with respect to CD spectra, iron binding and release, stability to proteolysis and heat stability. Deglycosylation was carried out at pH 6.0 on the iron-free form of lactoferrin, using an endoglycosidase preparation from Flavobacterium meningosepticum, comprising PNGase F and Endo F. Deglycosylation was rapid for human lactoferrin, being essentially complete within 12-24 hr. Only partial deglycosylation of bovine lactoferrin could be achieved under the same conditions, however, and this is attributed to the relative inaccessibility of at least one of the glycosylation sites. The CD spectra of native and deglycosylated human lactoferrins were found to be essentially identical in the range 250-350 nm, implying the same three dimensional structures. Both also bind iron in identical fashion; 2 Fe3+ ions are bound and binding is complete within 1 minute. The release of iron as the pH was lowered from 8.0 to 2.0 also showed no significant difference, the pH at which 50% release had taken place being 3.2 and 3.0 respectively for native and deglycosylated proteins. Susceptibility to proteolytic digestion by bovine trypsin over a period of 24 hr showed similar fragmentation patterns and a similar time course for the reaction for both species. Iron binding ability as a function of temperature was used as a measure of heat stability; melting temperatures derived from these experiments were 64°C for native and 63°C for deglycosylated lactoferrin. Comparison of the three dimensional structures of glycosylated iron-lactoferrin with deglycosylated apo-lactoferrin are consistent with these results, showing only a small increase in flexibility near the glycosylation site, when the carbohydrate is removed. Conclusions are that the in vitro physicochemical properties of lactoferrin are unaffected by the presence or absence of its glycan chains. In vivo studies may be necessary to establish the importance, if any, of glycosylation.Item The bovine lactoferrin gene : defining the minimal promotor region : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry(Massey University, 1997) Johnson, Daniel JeremiahLactoferrin is an 80 kDa glycoprotein with two lobes, each of which bind a single iron atom. Originally isolated from milk, lactoferrin has since been identified in a variety of exocrine secretions and in the secretory granules of neutrophils. A number of functions have been proposed for lactoferrin, some of which are related to the capacity of the protein to bind iron tightly but reversibly. The proposed functions include iron transport in the gut, antimicrobial activity and modulation of the activity of the immune system. The synthesis of lactoferrin in the mammary gland is developmentally regulated with changes in protein concentration in milk being correlated with changes in lactoferrin mRNA in mammary tissue. Most species studies have identified high levels of lactoferrin during involution and late pregnancy into early lactation. The amounts of lactoferrin during the lactational phase are much lower, especially in bovine milk. The regulation of bovine lactoferrin expression was studied in the belief that knowledge of the factors influencing expression will provide insight into the function of lactoferrin in the mammary gland. A 2.5 kb fragment of bovine genomic DNA, including the region immediately upstream of the transcription start point, has been subcloned into luciferase reporter gene vectors. The 2.5 kb fragment has been sequenced and a number of putative response elements identified. Promoter activity was tested by transient expression in the human endometrial carcinoma cell line RL 95-2. 5'- and 3'- deletion analysis of the promoter was used to establish regions which confer transcriptional regulation and the minimal promoter region. A recent report on the sequence of the cDNA for caprine (goat) lactoferrin suggests that the transcription start point for the mRNA for this protein may be further upstream than that reported for the mRNA of bovine lactoferrin. In view of the high level of sequence identity between the two cDNA's in this region an attempt was made to reinvestigate the transcriptional start point for bovine lactoferrin using DNA footprinting.Item The responsiveness of the bovine lactoferrin promoter to cytokines and glucocorticoids : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1998) Allen, Kirsty AnnLactoferrin is an iron-binding protein found in many bodily secretions and in the secondary granules of polymorphonuclear leukocytes. While there are many proposed functions for the lactoferrin protein - e.g. for iron storage, antibacterial properties, or a role in inflammation, the specific function(s) of lactoferrin have yet to be elucidated. Evidence that lactoferrin may be involved in inflammation was observed by Harmon et al. (1976) where after the induction of bovine mammary infections, a significant increase in secreted lactoferrin protein was seen during the early phase of the infection. As this increase was during the period of the acute phase response, this suggested that lactoferrin, as was the case with other proteins induced during this time, may have a role in the inflammatory response. The bovine lactoferrin (bLf) promoter contains many putative binding sites for inflammatory modulators, which suggests that the increases in lactoferrin seen during inflammation may be due to activation of lactoferrin gene transcription by these specifically-induced transcription factors. Substantiation of this suggestion would provide further evidence for a specific role for lactoferrin during inflammation. To investigate the cytokine-responsiveness of the bLf promoter, constructs corresponding to various lengths of the putative bLf promoter were linked to the luciferase reporter gene and introduced, by transient transfection, into RL95-2 human endometrial carcinoma cells. Cytokines, glucocorticoids or expression vectors for transcription factors were added to the cells, or potential 'masking' factors in the media such as phenol red or insulin were removed. The luciferase activity of the transfected cells was monitored for significant variation from the basal levels. The addition of cytokines with or without phenol red or insulin did not cause any significant changes in bLF promoter activity. In phenol red-free media, increases in luciferase reporter gene activity were observed after the co-transfection of an expression vector for NF-IL6, the addition of dexamethasone and also the addition of dexamethasone together with the co-transfection of a glucocorticoid receptor expression vector. These data provided evidence that lactoferrin transcription may be induced by inflammatory factors which support the suggestion that lactoferrin has a role in the inflammation process.Item The development of an enzyme-linked immunosorbent assay for bovine lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University(Massey University, 1988) Mock, Michele KayMethods were established for the estimation of bovine lactoferrin by enzyme-linked immunosorbent assay (ELISA) on microtitre plates and nitrocellulose (dot assay). Mfinity-puri:fied antibodies to bovine lactoferrin were prepared and conjugated to horseradish peroxidase by the periodate method. Conjugates with enzymatic and immunological activity had an apparent molecular weight of 65 or 95 kd. Four methods of ELISA on microtitre plates and three dot assays were developed. ii Differences between the seven assays could be attributed to the absorption capacity of the solid phase and the types of conjugates and substrates used. The range of the two successful quantitative assays were 3-lOOOng lactoferrin/ml (sandwich) and 60-8000ng lactoferrin/ml (competitive), while the qualitative dot assays had a range of 1-lOOJ.lg lactoferrin/ml. More replicates would be required to reduce variability. Results from these assays generally corresponded to results from ROCKET electrophoresis. Dot assay on nitrocellulose has a greater potential for reproducible and quantitative assays than assay on microtitre plates, because of the greater adsorption capacity of the nitrocellulose. In addition, the dot assays are faster and lend themselves to more applications than either ROCKET electrophoresis or ELISA on microtitre plates. The ELISA developed in this project appear to be the first alternatives to radial immunodiffusion and ROCKET electrophoresis for the measurement of bovine lactoferrin.Item Expression and characterisation of the n-terminal half of human lactoferrin : [t]his thesis is submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry(Massey University, 1993) Day, Catherine LouiseLactoferrin is an 80 kDa iron binding protein which is found in human milk and other exocrine solutions. Each molecule contains two metal binding sites which each bind a single iron atom with high affinity. The properties of the two sites are slightly different. In an attempt to more fully understand the nature of these differences a construct for the high level expression of the N-terminal half of human lactoferrin (LfN) has been constructed and protein expressed from this construct has been purified and characterised. Characterisation of the recombinant protein has shown that the signal peptide is correctly removed from LfN and that an N-linked carbohydrate moiety is added to LfN. LfN has been shown to bind one iron atom and the spectral properties are very similar to those of Fe2Lf. The most significant difference between hLf and LfN is in the pH stability of iron binding. Iron is released from LfN 2 pH units higher than from hLf. In an attempt to understand the bases for this difference a structural analysis of LfN was initiated. Using deglycosylated protein high quality crystals of both iron free and iron saturated LfN have been grown. The structures of both FeLfN and ApoLfN have both been solved by molecular replacement using the coordinates from the N-lobe of Fe2Lf as the starting model. The structure of FeLfN has been refined using data between 8.0 and 2.0 Å. The current model has good geometry and is believed to accurately represent the structure of FeLfN. The structure of FeLfN provides the highest resolution and most accurate structure of a member of the transferrin family. Analysis of the structure has shown that the folding pattern and the environment of the iron atom in FeLfN are very similar to the N-lobe of Fe2Lf although several differences exist. Most of the differences seen are due to the absence of the C-lobe and the rearrangement of residues 315 - 327. The altered conformation of residues 315 - 327 and the changes in the solvent accessibility to other residues are believed to be responsible for the different iron binding and release properties of LfN. Although the structure of ApoLfN is not complete analysis of this structure has shown that unlike the N-lobe of intact apo hLf the domains are closed in ApoLfN. The structure of ApoLfN is very similar to that of FeLfN even though the crystal packing is quite different. In addition although the protein was believed to be iron free there is some density in the iron site which is unaccounted for at present. This study continues. Several mutants of LfN have also been created. These mutants have shown that the carbohydrate groups attached to lactoferrin probably have a role in folding and secretion of lactoferrin by BHK cells. Several mutants involving changes to residues involved in metal and anion binding have also been created. These mutants have helped us begin to define the changes responsible for preventing iron binding in the C-lobe of melanotransferrin. In addition the role of arginine 121 has been investigated however further analysis of these mutants is required before the structural changes responsible for the different properties can be defined.Item Studies on lactoferrin, a metal binding protein in human milk : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University(Massey University, 1979) Plowman, Jeffrey ErnestLactoferrin (Lf), isolated from human colostrum, has been complexed with a variety of transition metal ions. In addition to binding two iron(III) or copper(II) ions it strongly and specifically binds two cobalt(III), chromium(III) and manganese(III) ions, in the presence of bicarbonate. Such strong, specific binding of copper(II) to lactoferrin will not occur in the absence of bicarbonate, instead only a weak interaction is observed. Lactoferrin has also been shown to weakly bind manganese(II), cobalt(II), nickel(II), zinc(II), cadmium(II), lead(II) and mercury(II), though manganese(II) will undergo aerial oxidation in the presence of the protein and bind as manganese(III). Those metal ion protein complexes have been examined using the techniques of fluorescence, electronic, e.s.r. and Resonance Raman spectroscopy. The close similarity between the spectra of the complexes Fe2Lf, Cu2Lf, Co2Lf, Mn2Lf and Cr2Lf with those of transferrin and ovotransferrin reinforce the postulate that the metal binding sites in all three proteins are similar. The sites in all three proteins are essentially rhombic in character and contain 2-3 tyrosyl residues and at least one histidyl residue. A distinct heterogeneity is evident from the e.s.r. spectrum of Cr2Lf and metal ion replacement studies indicate that chromium in one site is more labile than the other. In addition to the Specific sites lactoferrin appears to have a number of non-specific sites on the outside of the protein capable of weakly coordinating metal ions such as chromium(III) and copper(II). Differences evident in the non-specific sites available to copper when manganese(III) is bound to lactoferrin, to those available when iron(III), cobalt(III) and chromium(III) are bound, suggest that the stronger binding of manganese(III) results from it inducing a different conformational change in the protein to those other metals and one that favours the higher stability of its metal-protein complex. A series of small molecular weight complexes of iron(III) with bi-, tri-, tetra- and hexadentate ligands, containing phenolate groups were prepared and examined by electronic, e.s.r. and Mössbauer spectroscopy. Complexes of copper(II) with nitrogen bases and chloro- and bromo-substituted phenolates were prepared and examined by electronic and e.s.r. spectroscopy. Results from these studies would favour the involvement of three tyrosyl residues and two cis histidyl residues in the metal-binding sites of lacto-ferrin and from studies on the copper complexes it seems likely that one histidyl and one tyrosyl are axially coordinated to the metal. Evidence from a single crystal x-ray structure is presented which would favour the interaction of bicarbonate ion with iron(III) via a solvent (water) molecule.Item Expression, purification and characterisation of antimicrobial peptides of human and bovine lactoferrins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand(Massey University, 1999) Rallabhandi, Venkata Siva Prasad SarmaLactoferrin (Lf). a basic. ~80 kDa iron-binding glycoprotein, is a member of the transferrin family. It is present in the milk and other secretory fluids of many, but not all, mammalian vertebrates. Several biological functions have been ascribed to this protein. These include bactcriostastis, modulation of the inflammatory response, iron nutrition, a role as an anti-oxidant and regulation of myelopoiesis. Full-length human lactoferrin has been expressed in BHK cells, many strains of Aspergillus and with limited success in Saccharomyces cerevisiae. The main aim at the start of this project was to express full-length human lactoferrin (hLF) cDNA in the yeast Kluyveromyces lactis on whey-based media. Yeasts of the genus Kluyveromyces have been used for many years in the food industry and are classified as "Generally Regarded As Safe" (GRAS) organisms. K. lactis has impressive secretion capabilities and can grow on whey-based media (which is abundantly available in New Zealand). Attempts were made to sub-clone full-length hLF cDNA into the K. lactis vector, pEPS1 and the S. cerevisiae vector, pYEXS1 and to express the protein. The establishment of stable insert-carrying constructs of these yeast vectors in E. coli turned out to be an unattainable goal. Direct transformation of the ligation mix into K. lactis produced transformants, which secreted human lactoferrin protein products into the media as assessed by the lactoferrin ELISA assay. The secretion of hLF protein products by recombinant K. lactis continued for few generations, but gradually stopped. There are no known reports on the use of these vectors for the expression of any mammalian proteins in yeast. Lactoferrin has antimicrobial activity against a broad range of Gram-negative and Gram-positive bacteria and against fungi. Originally, the antimicrobial effect of lactoferrin was attributed to its ability to tightly sequester two atoms of iron and hence inhibit microbial growth through nutritional deprivation of iron. Recently, an N-terminal peptide called lactoferricin, isolated from the acid-pepsin hydrolysate of lactoferrin was shown to have greater antimicrobial activity than the intact protein. Currently, the only way to obtain native lactoferricins is to isolate the peptides from the acid pepsin-hydrolysate of lactoferrin, which gives very low yields, or to synthesise them by protein chemical methods, which is very expensive on a large scale. So, heterologous expression of both human and bovine lactoferricins in E. coli was attempted in this study. Synthetic DNA fragments encoding both human and bovine lactoferricins and 3'-end variants of these fragments were sub-cloned into E. coli expression vectors. pPROEXHTa, pET-15b and pGEX-4T1. The constructs were designed to express lactoferricins either as poly-His- or as GST-fusion proteins. In all cases the fusion proteins were expressed as inclusion bodies. The inclusion bodies were urea solubilised and purified on appropriate affinity resins. However, none of the recombinant proteins remained soluble after the urea was removed and therefore could not be further characterised. A similar situation was encountered by other investigators who attempted to express cationic peptides in E. coli. Both lactoferrin and lactoferricin have been shown to bind to the lipid A portion of the bacterial cell wall lipopolysaccharide (LPS), induce the release of LPS and kill the bacteria. In this work, five different E. coli strains were shown to have different susceptibility to native lactoferricin B. Transmission electron microscopy studies of the E. coli strains treated with lactoferricin B revealed considerable differences in their membrane ultrastructure. The most susceptible E. coli strain showed a direct outer membrane dislocation and effect on the cytoplasmic contents. A relatively resistant E. coli strain showed an initial formation of 'membrane blisters'. However, after a long exposure to lactoferricin B, a few cells of this strain showed an outer membrane dislocation and effect on the cytoplasmic contents. The formation of 'membrane blisters' might allow the relatively resistant strain of E. coli to reduce the lethal action of lactoferricin B.