Glycosylation of Human Milk Lactoferrin Exhibits Dynamic Changes During Early Lactation Enhancing Its Role in Pathogenic Bacteria-Host Interactions
2012; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês
10.1074/mcp.m111.015248
ISSN1535-9484
AutoresMariana Barboza, Janneth Pinzon, Saumya Wickramasinghe, John W. Froehlich, Isabelle Moeller, Jennifer T. Smilowitz, L. Renee Ruhaak, Jincui Huang, Bo Lönnerdal, J. Bruce German, Juan F. Medrano, Bart C. Weimer, Carlito B. Lebrilla,
Tópico(s)Pediatric Hepatobiliary Diseases and Treatments
ResumoHuman milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association. Human milk lactoferrin (hmLF) is the most abundant glycoprotein present in human milk and displays a broad range of protective functions in the gut of newborn infants. hmLF is N-glycosylated, but little is known about the lactation stage-related development of the glycosylation phenotype. hmLF glycosylation from milk samples from five donors during the first 10 weeks of lactation was assessed and observed to be more diverse than previously reported. During this period dynamic changes in glycosylation were observed corresponding to a decrease in glycosylation in the second week followed by an increase in total glycosylation as well as higher order fucosylation thereafter. Gene expression analysis was performed in milk somatic cells from a sixth subject. It was found that fucosyltransferase expression increased during entire period, whereas expression of genes for the oligosaccharyl transferase complex decreased in the second week. The effect of hmLF glycosylation was examined for the protein's ability to affect bacterial binding to epithelial cells. hmLF significantly inhibited pathogen adhesion and purified hmLF glycans significantly reduced Salmonella invasion of colonic epithelial cells to levels associated with non-invasive deletion mutants. This study indicates that hmLF glycosylation is tightly regulated by gene expression and that glyco-variation is involved in modulating pathogen association. Human milk constitutes the first source of nutrients for the newborn infant, but it has also evolved to endow several key physiological advantages to the neonate. Other than to provide the neonate with energy and amino acid building blocks, proteins possess a wide range of biological activities that promote the normal development and maturation of specific organs in the newborn, specifically, the functions of the gut mucosa and the growth of gut microbiota (1Lönnerdal B. Lien E.L. Nutritional and physiologic significance of alpha-lactalbumin in infants.Nutr. Rev. 2003; 61: 295-305Crossref PubMed Scopus (137) Google Scholar). Human milk proteins also display a protective effect against infectious diseases via antimicrobial and immuno-modulatory activities that confer passive immunity to the breast-fed infant (1Lönnerdal B. Lien E.L. Nutritional and physiologic significance of alpha-lactalbumin in infants.Nutr. Rev. 2003; 61: 295-305Crossref PubMed Scopus (137) Google Scholar, 2Kaufman D.A. Lactoferrin supplementation to prevent nosocomial infections in preterm infants.JAMA. 2009; 302: 1467-1468Crossref PubMed Scopus (10) Google Scholar, 3Venkatesh M.P. Rong L. Human recombinant lactoferrin acts synergistically with antimicrobials commonly used in neonatal practice against coagulase-negative staphylococci and Candida albicans causing neonatal sepsis.J. Med. Microbiol. 2008; 57: 1113-1121Crossref PubMed Scopus (53) Google Scholar). Many of these proteins are post-translationally modified and the possible roles of such modifications in mediating demonstrated bioactivities are largely unexplored. Lactoferrin (LF) 1The abbreviations used are:LFLactoferrinBSSLbile-salt stimulated lipaseDHB2,5-dihydroxybenzoic acidFUTfucosyltransferasehmLFhuman milk lactoferrinIRMPDinfrared multiphoton dissociationMWCOmolecular weight cut offOSToligosaccharide transferasePNGase Fpeptide N-glycosidase FRPKMreads per kilo base per million mapped readsSEMstandard error of the meanSWIFTstored-waveform inverse Fourier transformTFAtrifluoroacetic acidFTICRFourier Transform Ion Cyclotron Resonance. 1The abbreviations used are:LFLactoferrinBSSLbile-salt stimulated lipaseDHB2,5-dihydroxybenzoic acidFUTfucosyltransferasehmLFhuman milk lactoferrinIRMPDinfrared multiphoton dissociationMWCOmolecular weight cut offOSToligosaccharide transferasePNGase Fpeptide N-glycosidase FRPKMreads per kilo base per million mapped readsSEMstandard error of the meanSWIFTstored-waveform inverse Fourier transformTFAtrifluoroacetic acidFTICRFourier Transform Ion Cyclotron Resonance. is an iron-binding glycoprotein found in milk from most species, but human milk LF (hmLF) is the most abundant glycoprotein present in colostrum and mature milk (6–8 mg/ml and 2–4 mg/ml, respectively) (1Lönnerdal B. Lien E.L. Nutritional and physiologic significance of alpha-lactalbumin in infants.Nutr. Rev. 2003; 61: 295-305Crossref PubMed Scopus (137) Google Scholar, 4Garcia-Montoya I.A. Cendon T.S. Arevalo-Gallegos S. Rascon-Cruz Q. Lactoferrin a multiple bioactive protein: An overview.Biochim. Biophys. Acta. 2011; 1820: 226-236Crossref PubMed Scopus (312) Google Scholar). The presence of glycans on hmLF is long known (5Spik G. Strecker G. Fournet B. Bouquelet S. Montreuil J. Dorland L. van Halbeek H. Vliegenthart J.F. Primary structure of the glycans from human lactotransferrin.Eur. J. Biochem. 1982; 121: 413-419Crossref PubMed Scopus (159) Google Scholar), but so far, the only role identified is to protect the molecule from proteolysis (6van Veen H.A. Geerts M.E. van Berkel P.H. Nuijens J.H. The role of N-linked glycosylation in the protection of human and bovine lactoferrin against tryptic proteolysis.Eur. J. Biochem. 2004; 271: 678-684Crossref PubMed Scopus (95) Google Scholar). 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Despite numerous studies establishing the variation in total protein concentration as well as composition of human milk, there has been little effort characterizing the variation in glycosylation of milk glycoproteins over the course of lactation. Only one protein, namely bile-salt stimulated lipase (BSSL) was shown to have a dynamic glycosylation pattern over the course of lactation (20Landberg E. Huang Y. Strömqvist M. Mechref Y. Hansson L. Lundblad A. Novotny M.V. Påhlsson P. Changes in glycosylation of human bile-salt-stimulated lipase during lactation.Arch. Biochem. Biophys. 2000; 377: 246-254Crossref PubMed Scopus (46) Google Scholar). The biological significance of these findings is, however, so far unclear and unexplored. Given the central role of lactoferrin in infant development and health and its status as the most abundant glycoprotein in milk, we examined the changes in glycosylation during the first months of lactation with the hypothesis that glycan variation is common over the course of lactation as a mechanism to block pathogen association during breastfeeding. hmLF binds several pathogenic Gram-positive (21Qiu J. Hendrixson D.R. Baker E.N. Murphy T.F. St Geme 3rd, J.W. Plaut A.G. Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12641-12646Crossref PubMed Scopus (116) Google Scholar, 22Hammerschmidt S. Bethe G. Remane P.H. Chhatwal G.S. Identification of pneumococcal surface protein A as a lactoferrin-binding protein of Streptococcus pneumoniae.Infect. Immun. 1999; 67: 1683-1687Crossref PubMed Google Scholar) and Gram-negative (23Ochoa T.J. Noguera-Obenza M. Ebel F. Guzman C.A. Gomez H.F. 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Yamauchi K. Wakabayashi H. Kawase K. Tomita M. Identification of the bactericidal domain of lactoferrin.Biochim. Biophys. Acta. 1992; 1121: 130-136Crossref PubMed Scopus (817) Google Scholar). In addition, hmLF may inhibit infections caused by viruses, yeast, fungi, parasites, and other eukaryotic microbes (27Jenssen H. Hancock R.E. Antimicrobial properties of lactoferrin.Biochimie. 2009; 91: 19-29Crossref PubMed Scopus (354) Google Scholar). However, to date, the role of glycosylation of hmLF in these antimicrobial, antiviral, antifungal and antiparasitic activities has not been elucidated. We determined the N-glycan profile of human milk lactoferrin by mass spectrometry analysis in individual samples of hmLF purified from five donors during the first 72 days of lactation. The expression of genes associated with glycosylation in milk somatic cells was assessed to evaluate the regulation of the dynamic glycosylation. The biological and/or functional significance of glycans found in hmLF was determined using in vitro studies of host-microbe interactions with colonic epithelial cells and gastrointestinal bacterial pathogens in the presence of hmLF glycoforms and released N-glycans. A purified human milk lactoferrin standard was obtained from Sigma Aldrich (St. Louis, MO), Heparin-Sepharose 6 fast flow was purchased from GE Healthcare (Pittsburgh, PA), and 10 ml econopack columns were purchased from Bio-Rad (Richmond, CA). Glycerol free peptide N-glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA). α-1–3/4 fucosidase (from Xantomonas sp.) was obtained from Calbiochem (San Diego, CA), and β-1–4 galactosidase from Glyco (Novato, CA). Recombinant α-2–3/6 sialidase was a kind gift from Dr. David Mills (Department of Viticulture and Enology, UC Davis). Solid-phase-extraction graphitized-carbon and C8 cartridges were purchased from Glygen corporation (Columbia, MD) and Supelco (Bellefonte, PA), respectively and Microcon centrifugal filter devices (ultracel YM-10) were from Millipore Corporation (Bedford, MA). Acetonitrile and trifluoroacetic acid were ACS quality or higher. Samples were donated by five healthy women from Reno, NV, who gave birth to term infants (> 38 weeks). Overall, human milk samples collected on days 1, 5, 10, 15, 30, 44, 58, and 72 postpartum were interrogated in this study. All milk samples were manually expressed and immediately frozen. Samples were then transferred to a −80 °C freezer within 3 h and stored until analysis. LF purification from individual milk samples was performed in parallel following a procedure described by Lonnerdal et al. (37Carlsson J. Porath J. Lönnerdal B. Isolation of lactoferrin from human milk by metal-chelate affinity chromatography.FEBS Lett. 1977; 75: 89-92Crossref PubMed Scopus (85) Google Scholar) with slight modifications, as follows. Briefly, whole human milk samples (0.5 ml) were centrifuged at max speed, for 30 min, at 4 °C. The lower aqueous phase was recovered in a new tube and a CaCl2 solution (pH 4.6) was added to a final concentration of 60 mm. The mixture was incubated 1 h at room temperature (∼25 °C), and further centrifuged at 6750 × g for 20 min at room temperature. Empty columns were packed with 1 ml of heparin-Sepharose resin and equilibrated with 50 mm Tris HCl pH 8.0 (running buffer). The whey fractions obtained were loaded onto the columns and the flow-through was collected and reloaded onto the column twice. Columns were closed and the samples were allowed to interact with the resin for 3 h at room temperature. Upon washing with 15 ml of running buffer, weakly bound proteins were eluted using five column volumes of 50 mm Tris HCl pH 8, 0.3 m NaCl (EBI), and 1 ml fractions were collected. LF bound to heparin-Sepharose and was eluted with five column volumes of 50 mm Tris HCl pH 8, 1 m NaCl (EBII). Fractions were collected, dialyzed against 10 mm ammonium bicarbonate, concentrated and stored at −20 °C. Protein concentration was determined using the Bradford assay and 5 μl aliquots were assayed by SDS-PAGE. Commercially available LF and LF purified from single samples from all donors during the course of lactation (20 μg) were reduced and alkylated in 50 mm NH4CO3. PNGase F (1 μl, or 500 NEB units) was added to all samples and glycans were released by incubation at 37 °C for 16 h. Released glycans were purified by solid phase extraction using porous graphitized carbon cartridges. The cartridges were conditioned with three volumes of deionized water, followed by three volumes of 80% acetonitrile in 0.1% aqueous trifluoroacetic acid (v/v) and another three volumes of deionized water. The oligosaccharide samples were loaded onto the cartridge, incubated for 10 min at room temperature, and washed with three volumes of deionized water. Elution was performed to fractionate the oligosaccharides mixture into two fractions previous to MS analysis in order to minimize suppression. Glycans were eluted with three volumes of 20% acetonitrile in water (v/v), followed by three volumes of 40% acetonitrile, 0.1% trifluoroacetic acid in water (v/v) and dried in vacuo. Glycans were reconstituted in 10 μl of deionized water. All mass spectra were acquired on an HiRes MALDI-FTICR MS instrument with an external MALDI source, a 355-nm pulsed Nd:YAG laser, a quadrupole ion guide, and a 7.0 Tesla superconducting magnet (IonSpec, Irvine, CA). The analyte-matrix deposit was prepared with 1 μl of sample being spotted on the MALDI plate, followed by the addition of 0.1 μl of 0.1 mm NaCl as a dopant and 1 μl of 0.4 m DHB. The spots were allowed to dry under vacuum before analysis. For detection, ion excitation was performed through an arbitrary waveform with amplitude of 150.0 V (base to peak) at a rate of 2 MHz for a scan range of m/z 216–4500 and 1024K data points. Five acquisition scans were performed on each sample in the positive ion mode. The instrument was externally calibrated with a malto-oligosaccharide mixture, and glycans were identified by accurate mass with a mass tolerance < 5 ppm. For MS/MS analysis, individual glycan ions were selected within the ICR cell using stored-waveform inverse Fourier transform (SWIFT) isolation prior to collision-induced-dissociation (CID), and infrared multiphoton dissociation (IRMPD). For IRMPD experiments, the infrared radiation was supplied by a 10.6 μm, 20 W CO2 laser (Parallax Laser, Inc., Waltham, MA). Fragmentation was optimized by varying the IRMPD laser pulse time between 500 and 1500 ms. Irradiation time was increased until the majority of the precursor ion was dissociated. Sialic acid-, fucose-, and galactose-free hmLF glycoforms were created by treatment of standard hmLF with the corresponding exoglycosidases, followed by ultrafiltration. For each treatment 2 mg of hmLF was reconstituted in 1 ml of appropriate exoglycosidase digestion buffer. Digestion with α-2–3/6-sialidase (recombinant, gift from Dr. David Mills, UC Davis) was carried out in 50 mm ammonium phosphate pH 6, treatment with α-1–3/4-fucosidase (Calbiochem, San Diego, CA) in 50 mm sodium phosphate pH 5.5, and digestion with β-1–4-galactosidase (Glyco, Novato, CA) was performed in 50 mm Tris-HCl pH 7. The corresponding enzymes (3 μl) were added to each reaction tube and incubated with agitation for 24 h at 37 °C in a dry oven. After digestion, the samples were frozen to inactivate the enzymes and released monosaccharides were removed from hmLF glycoforms by ultrafiltration using deionized water and Microcon centrifugal devices with a MWCO of 10 kDa. LF glycoforms were recovered in 1 ml of water and protein concentration was determined using Bradford. Free hmLF N-glycans were prepared from 2 mg of reduced and alkylated LF with PNGase F as described above and subsequently separated from the protein by solid phase extraction using a C8 cartridge conditioned with six volumes of acetonitrile followed by six volumes of water. Sample was loaded and glycans were recovered in 9 ml of water, followed by drying in vacuo. Fresh milk samples were obtained from a healthy female on days 4, 15, 30, and 40 postpartum who gave birth to a term infant (> 38 weeks). In the early morning period, the donor manually pumped one breast until emptied into a collection bag, and immediately delivered on cold-packs to the lab for processing. Samples were divided into two aliquots of ∼20 ml for oligosaccharide profile analysis and RNA extraction from somatic cells. The Institutional Review Board of University of California, Davis, approved the project. The research was conducted in accordance with the ethical standards outlined in the Helsinki Declaration, with all participants providing written informed consent. Somatic cells were pelleted by adding 50 μl of 0.5 m EDTA to 20 ml of fresh milk and centrifuged at 1800 rpm at 4 °C for 10 min. The pellet of cells was washed with 10 ml of phosphate-buffered saline at pH 7.2 and 10 μl of 0.5 m EDTA (final conc. 0.5 mm) and filtered through sterile cheesecloth to remove any debris. The cells were then centrifuged again at 1800 rpm, 4 °C for 10 min. The supernatant was decanted and RNA was extracted from the milk somatic cell pellet using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. RNA was quantified by an ND-1000 spectrophotometer (Fisher Thermo, Wilmington, MA) and the quality and integrity was assessed by the spectrophotometer 260/280 ratio, gel electrophoresis and by capillary electrophoresis with an Experion bio-analyzer (Bio-Rad, Hercules, CA). Gene expression analysis was conducted on fresh milk samples collected on days 4, 15, 30, and 40 postpartum by RNA sequencing (RNA-Seq). Messenger RNA was isolated and purified using RNA-Seq sample preparation Kit (Illumina, San Diego, CA and NuGen, San Carlos, CA). Subsequently, mRNA was fragmented to ∼200 bp fragments and first and second strand cDNA were synthesized, followed by end repair and adapter ligation. The fragments were purified and sequenced at the UC Davis Genome Center DNA Technologies Core Facility using the Illumina Genome Analyzer (GAII). Short sequence reads of 36–40 bp were assembled, and analyzed in RNA-Seq and expression analysis application of CLC Genomics workbench 3.7 (CLC Bio, Aarhus, Denmark). Human Genome, GRCh37.1 (http://www.ncbi.nlm.nih.gov/genome/guide/human/index.html) was utilized as the reference genome for the assembly. Data were normalized by calculating the "reads per kilo base per million mapped reads" (RPKM) for each gene (38Mortazavi A. Williams B.A. McCue K. Schaeffer L. Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq.Nat. Methods. 2008; 5: 621-628Crossref PubMed Scopus (9884) Google Scholar) and annotated with NCBI human genome assembly (35,489 unique genes). Intestinal epithelial cells (Caco-2; ATCC HTB-37) were grown as per the manufacturer's instructions in T-25 flasks at 37 °C with 5% CO2. Subsequently, for compound treatment, cells were seeded to a density of 105 cells/cm2 in a 96-well plate using Dulbecco's modified Eagle's medium/High Modified (Thermo Scientific, Rockford, IL) with 16.6% fetal bovine serum (FBS) (HyClone Laboratories, Logan, UT), nonessential amino acids (Thermo Scientific), 10 mm 3-(N-morpholino)propanesulfonic acid (Sigma, St. Louis, MO), 10 mm TES (Sigma), 15 mm HEPES (Sigma), and 2 mm NaH2PO4 (Sigma). Cells were incubated at 37 °C in 5% CO2 atmosphere for 14-days post confluence to allow differentiation (39Ouwehand A.C. Salminen S. In vitro Adhesion Assays for Probiotics and their in vivo Relevance: A Review.Microbial Ecology in Health & Disease. 2003; 15: 175-184Crossref Scopus (111) Google Scholar). The epithelial cells were washed once with 150 μl of PBS just prior to the treatment with hmLF preparations and bacterial addition. Prior to the adhesion assay, each bacterial culture was thawed from −70 °C stock cultures, transferred twice after growth for 16–18 h at 37 °C shaking at 250 rpm, and collected for use in the adhesion assay. Bacterial cells were collected from 15 ml of the respective medium after growth for 16 h, washed twice with an equal volume of PBS, and resuspended to an OD600 nm of 0.2 in Dulbecco's modified Eagle's medium/High Modified containing nonessential amino acids, 10 mm MOPS, 10 mm TES, 15 mm HEPES, and 2 mm NaH2PO4 without FBS. Each bacterial suspension (50 μl), was mixed with hmLF, hmLF glycoforms (500 μg/ml), or the purified N-glycans (250 μg/ml), mixed by vortexing for 1 min, and added to the washed, differentiated Caco-2 cells to a final multiplicity of infection of 1000. The Caco2 cells treated with bacteria and hmLF were incubated at 37 °C in an atmosphere containing 5% CO2 for 60 min to let the bacteria interact the epithelial cells. Supernatants were then aspirated and the Caco2 monolayer was washed thrice with 200 μl of Tyrodes buffer (pH 7.2) (40de Ridder L. Mareel M. Vakaet L. Adhesion of malignant and nonmalignant cells to cultured embryonic substrates.Cancer Res. 1975; 35: 3164-3171PubMed Google Scholar, 41Lominadze D.G. Saari J.T. Miller F.N. Catalfamo J.L. Justus D.E. Schuschke D.A. Platelet aggregation and adhesion during dietary copper deficiency in rats.Thromb. Haemost. 1996; 75: 630-634Crossref PubMed Scopus (30) Google Scholar) to remove nonadhered bacteria from the monolayer. Adhered and invaded bacterial counts were performed as described by Elsinghorst (42Elsinghorst E.A. Measurement of invasion by gentamicin resistance.Methods Enzymol. 1994; 236: 405-420Crossref PubMed Scopus (282) Google Scholar), except qPCR was used to determine the bacterial count, and that DNA extraction was performed using 50 μl of a commercial lysis buffer (AEX Chemunex, France) as described by Desai (43Desai P.T. Walsh M.K. Weimer B.C. Solid-phase capture of pathogenic bacteria by using gangliosides and detection with real-time PCR.Appl. Environ. Microbiol. 2008; 74: 2254-2258Crossref PubMed Scopus (16) Google Scholar). Quantitative bacterial analysis was performed using qPCR with a CFX 96 Real Time System (BioRad, Hercules, CA). Reactions were performed in a total volume of 25 μl containing 1 μl of cell lysate, 100 nm of PCR primers (Table I), and iQ SYBR Green Supermix (BioRad, Hercules CA) as per the manufacturer's instructions. The thermocycling parameters consisted of a denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation, annealing and extension at 95 °C for 15 s, 56 °C for 30 s, 72 °C for 30 s, respectively, and a final extension at 72 °C for 1 min. The amplified product was verified using melt curve analysis from 50 °C to 95 °C with a transition rate of 0.2 °C/s.Table IhmLF glycosylation changes over the course of lactation. A summary of the changes observed in each mother regarding total glycosylation content and levels of fucosylation are presented for each individual motherMother 1Mother 2Mother 3Mother 4Mother 5Days sampled1; 5; 10; 15; 301; 5; 15; 30; 58; 721; 5; 10; 15; 30; 44; 58; 723; 5; 10; 15; 30; 44; 57; 721; 5; 10; 15; 30; 44; 58; 72Glycan diversitydecreaseearly decrease, later recoveryconstantearly decrease, later recoveryconstantMonofucosylatedvariablelater decreasedecreaseconstantdecreaseDifucosylatedprogressive increaseprogressive increaseprogressive increaseprogressive increaseprogressive increaseTrifucosylatedvariablelater increaseconstantconstantlater increase Open table in a new tab Host-microbes interaction experiments were performed using three biological replicates. Bacterial analysis was performed using three technical replicates within each biological replicate. The number of adhered bacteria was determined by subtracting mean of invaded bacteria (B) from mean of total host associated bacteria (A). The error (ΔZ) was
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