Carlito B. Lebrilla Distinguished Professor

Transcription

1 Carlito B. Lebrilla Distinguished Professor Department of Chemistry Department of Biochemistry and Molecular Medicine (School of Medicine) University of California, Davis

2 Many functions of human milk Early studies on milk has focused on nutrition. Milk contains functional compounds that can have long term consequences to health. Components of milk Lactose (Glyco)Lipids Oligosaccharides (HMO) (Glyco)Proteins (Glyco)Peptides Metabolites Cells Minerals DNA Microbes RNA Micelles Milk is highly glycosylated Nutritio n Protection Infant Development Microbiota Development

3 Developed and employed tools for Oligosaccharide analysis Components of milk Lactose (Glyco)Lipids Oligosaccharides (HMO) (Glyco)Proteins (Glyco)Peptides Metabolites Cells Minerals DNA Microbes RNA Micelles Oligosaccharides are an analytical challenges: Developed many tools to characterize and quantitate glycosylation in tissue Milk is highly glycosylated Genomic Proteomic Glycomic

4 HMO library : at least two phenotypes to human milk Analysis >5000 milk samples Lewis b- Milk type Non Secretor Lewis b+ Milk type Secretor Fut2 Gene in epithelial cells Davis et al Manuscript Wu et al. JPR 2012 Wu et al. JPR 2012 Overlay >100 chromatogram Method for determination highly reproducible.

5 Antigens in HMO similar to blood type antigens Based on the analysis of milk, structures yielded biosynthetic pathways. Human milk oligosaccharides are similar to blood group determinants HMOs include many antigens and receptors Wu et al. J. Proteome Res. 2010, Wu et al. J. Proteome Res Binding site to pathogens

6 10uL of human milk LC-MS profile of HMO A library of HMO structures produced to understands role of human milk oligosaccharide Two methods for Analysis nanolc chip QTOF MS UPLC QQQ MS Totten et al. J. Proteome Res. 2012

7 Like Counting Hippopotamuses in river of crocodiles Count the Hippo in the water (UPLC-QQQ MS) May mistaken crocodiles for Hippo Hard to to tell the size of the hippo Hard to tell one hippo from another Hippo out of the water (NanoLC - QTOF MS) Convince hippo to leave the water Not all the hippos may leave No mistaking hippo for crocs Can tell hippos apart

8 Two methods for profiling HMO UPLC QQQ MS NanoLC - TOF MS UPLC QQQ MS Absolute quantitation (mg/ml) No enrichment Less specificity High throughput >200 samples/day NanoLC MS TOF Quantitation in ion counts Enrichment necessary to quantitate Better Specificity More compounds >300 Low Speed >24 samples/day

9 Methods for profiling HMO MALDI Probe MALDI MS (No isomer separation) Separation HPLC UV 1 hour analysis Widely available, Previous standard Absolute quantitation <30 compounds CE Fluorescence <1 minute analysis Absolute quantitation <30 compounds HPLC-QTOF MS 1 hour analysis Relative quantitation >300 compounds Discovery UPLC QQQ MS 10 minute analysis Absolute quantitation < 30 distinct compounds (separate by class) Requires chemical labeling Different reactivities Completeness of reactions (not 100%) limits dynamic range Requires commercial standards Expensive Very impure Variations in retention times Compounds misidentified Difficult to separate class Requires chemical labeling Different reactivities Completeness of reactions (not 100%) Requires commercial standards Expensive Very impure Variations in retention times Compounds misidentified Requires chemical reduction Differential ionization will yield different responses Requires standards

10 Factors that affect the abundances of HMO UC Davis Cohort Jennifer Smilowitz, J. Bruce German Sex of infant Age of Mother Number of children Gestational Diabetes Days postpartum Term Delivery Secretor Status Diet Absolute quantitation using MRM (LCQQQ) shows: Decrease in absolute abundances during lactation. Decrease in abundances of fucosylated structures. Decrease in abundances of sialylated structures Secretors have more HMO, Fucosylation, less sialylation Xu et al J Nutrition 2016 UPLC QQQ MS

11 MAL-ED:Total HMO Decreases Throughout Lactation Lactation Month Country United States Brazil Peru South Africa India Bangladesh Error bars given as standard deviation Order of country (left to right) decreasing GDP (per capita, based on 2014 United Nations rankings) Note that United States, Peru, South Africa, and Bangladesh have a month 0 time point Note that United States has no month 5

12 Diet affects HMO production The affect of large diet change on HMO was observed in studies in The Gambia. A swing of around 5 kg/year, mostly lost from stored body fat. Food sources begin to deplete in wet season, food becomes less available. Heavier work load, expending more energy than taking in.

13 David Mills Most infants rich in Bifidobacterium. Mothers lactating through wet season produce less HMO. Diet affects HMO production Nearly all Infants rich in Bifidobacterium Angela Zivkovic Production of HMO during birth and lactating season. HMO decreased during wet hunger season Davis et al Under Revision 2016

14 Diet affects HMO production Angela Zivkovic Structures correlated with child morbidity Infants with sick days had less LNFP1, more LNT. Mother self reported. Sick Any day with illness (cold, diarrhea, etc.) Not Sick No illness days reported.

15 Gates Fund Project on Environmental Entheropathy Sample Preparation/Extraction Project Sub-Project Description Sample Type # of Samples HMOs Glycopeptides Peptides Protein Measurement N-glycans LCNI-5/ Malawi Lactation/ Davis Healthy vs Stunted, extreme cases Timepoints (2-347 days) Control Study Milk X X X X Feces 54 X X (free) Paired Milk test set 107 X Paired Feces 107 X X X (free) Gates/BMMI MAL-ED -India -Peru -South Africa -Brazil -Bangladesh Timepoints (0-6 Months) Phenotypic bins of LAZ scores (HH, HR, SG, SS) Milk 516 X X X (n=92) Phenotypically Binned 280 X X Non-binned 570 X X ilins Dyad/ Malawi 3 nutrient suppl 3 Lactation Timepoints (1,3,6) Healthy Vs Severely Stunted Milk 259 (263 actual) X X Stunted/Non-stunted/ extreme cases 536 (553 actual) X X Received Processing (extraction + instrument run) Data Analysis Completed Jeff Gordon J. Bruce German David Mills Daniela Barile

16 Large Multisite Study on HMO and Stunting < 3 > 0 < 2 > 0 -Mothers of healthy infants had higher levels of total, fucosylated, and sialylated HMOs -Non-secretors mothers of stunted infants had significantly lower levels of HMO Charbonneau et al Cell 2016

17 Specific structures correlate with stunting Absolute abundances 3FL p-lnh 3 SL FL p-lnh 'SL Healthy Severely Stunted Healthy Severely Stunted Healthy Severely Stunted Month 1 Month 3 3FL P = FL P = LAZ bin LNFP II LNFP II P = P = a 5130a LAZ bin P = P = p-lnh 3120 P = P = LAZ bin 3 SL P = SL P = Month 6 3FL P = MFLNH III+MFLNH I P = * LNFP II P = TFLNH P = a P = LDFT P = LSTc P = P = SL P = LSTa P =

18 Secretor Status - a factor in interaction with gut microbiota Secretor B+ Milk Type Glucose (Glc) 6 Galactose (Gal) 4 N-Acetylglucosamine (GlcNAc) 3 Fucose (Fuc) 2 N-Acetylneuraminic Acid (Neu5Ac) B- Milk Type Secretor is more than just α(1,2)fucose Non-Secretor Charbonneau et al Nature 2016

19 Distribution of Lewis B+/B- milk Katie Hinde (U. Arizona) Andrew Prentice (U. Cambridge) J. Bruce German (UC Davis) MAL-ED (BMGF) Jeff Gordon ILYNS (BMGF) Jeff Gordon Combined > 1000 samples Map of Secretor distribution based on mother s milk. Davis et al. - Manuscript

20 Distribution of Lewis B+/B- milk Africa high levels of non secretors Out of Africa model shows nonsecretors diminishing. Nature 2016 US secretor more representative of Europe South America Indigenous have little or no non-secretors. Isolated tribes in South America Davis et al. - Manuscript

21 Cholera Outbreaks Correlate with High Number of Non-secretors Local diseases select secretor types Cholera could affect infants more strongly than adults. Cholera response affected by blood types. Fleckenstein Am J. Trop Med

22 HMO Found in other bodily fluids Mother Milk Blood Infant Blood Urine Feces HMO, modified HMO HMO HMO, HMO fragments, N-glycans J Bruce German Mark Underwood Infant Blood Mother Blood

23 HMO Found in Infant Blood Blood of infants yield HMO and BMO. HMO found in blood of breast fed infants. Breast + formula Exclusively formula BMO found in blood of formula fed infants. Distinguish between breast fed and formula fed infants. Ruhaak et al. Anal Bioanal Chem 2014

24 HMO Found in Mother s blood HMOs found in blood of lactating women ~1 ug/ml HMO in blood. BMO primarily found in blood of Men and nonlactating women. x 3 Some HMO found in blood of men and non-lactating women. Xu et al. Manuscript

25 HMOs are prebiotic David Mills Consumption patterns of B. infantis strain HMO Bifido longhum sp infantis Enzymes found in B infantis cleave Sialidases Sela et al JBC 2011 Fucosidases Sela et al AEM 2012 Hexosaminidases Garrido et al Anaerobe 2012 Galactosidases Garrido et al Food Micro 2012 Surface Binding Proteins Garrido et al PLoS One 2011 Strum et al An Chem 2012

26 HMO in feces increase until commensals are established HMO builds up in feces until a sufficient population of saccharolytic bacteria populates the gut. The abundance of HMO in feces decreases significantly when this condition is met. DeLeoz et al. J Proteome Res 2014 Bifido longhum sp infantis

27 Feces contain glycans from glycoproteins

28 Feces contain glycans from glycoproteins Digested HMO Digested N-glycans Tandem MS (MS/MS) used for structural elucidation. Structures correspond to digested HMO and digested N-glycans. HMO and Glycoproteins sources of prebiotic! Davis et al. Molec Cellular Proteomic 2016

29 Feces contain glycans digested by B. Infantis enzymes Breast milk B-galactosidase BLNG_00015 Feces Identify structures Digestion of HMO with expressed enzymes yielded the structures in feces. Collective enzymes in the gut biota produces feces products. BLNG_00015 PNGAse F EndoBI-1

30 LC-MS method for monosaccharide analysis Quantification of monosaccharides using 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization We can follow the degradation to the monosaccharides. New quantitation method mates well with HMO analysis. Current Monosaccharide analysis uses GC-MS, requires three days and ug for analysis. New method allows 100 samples/day, with nanograms. UPLC QQQ MS

31 Counts vs. Acquisition Time (min) Monosaccharide analysis in feces Dynamic MRM chromatogram of a fecal sample (P1A) Quantitation results of 22 fecal samples (reducing monosaccharides) x Man All Rha GlcNAc Xyl GalNAc Ar a x Counts vs. Acquisition Time (min) GlcA Gal Glu Fuc GalA Sample GlcNAc GalNAc All Ara Fuc Gal GalA Glc GlcA Man Rha Xyl P1A (μg/ml) Monosaccharides in feces show complementary (weaning) foods

32 HMO Glycoproteins feces increase are effective until pathogen commensals block are - established Lactoferrin Components of milk Lactose (Glyco)Lipids Oligosaccharides (HMO) (Glyco)Proteins (Glyco)Peptides Metabolites Cells Minerals DNA Microbes RNA Micelles Glycosylation in proteins interact with the microbiome. Function as pathogen block. New sources of prebiotic.

33 HMO Glycoproteins feces increase are effective until pathogen commensals block are - established Lactoferrin Bart Weimar Glycosylation on Lactoferrin varies during the lactation period. Glycans are rich in fucose and sialic acids. Specific glycoforms bloc, distinct bacteria Mariana Barboza, Molec. Cell Proteom. 2012

34 Lactoferrin Glycan Map of milk glycoproteins IgM IgG alpha-1-antitrypsin Systematic map of milk glycoproteins. Extensive site heterogeneity. Quantitate proteins and gycoforms. IgA Huang et al. - Revision

35 Selection of Peptides for Quantitation (IgG Fc region) Protein identification and peptide quantification Quantitate protein (absolute) Quantitate Glycopeptide (relative) IgG1 IgG2 IgG3 Normalize glycopeptide to protein abundance Q Hong et al Anal Chem 2014 IgG4 Peptide common in all the subclasses Peptides unique for each subclass N-glycosylation site

36 IgG tryptic Glycopeptide - Quantitation MRM with UPLC QQQ MS Glycosylation site Each glycoform can be monitored Each glycoform monitored in tryptic mixture.

37 Analytical Method for Proteins and Glycoproteins Multiple reaction monitoring Mass Spectrometry Rapid throughput (15 min/run) Sensitive 10 ul milk Absolute quantitation of proteins with glycosylation.

38 Abundance of Proteins among Countries Month 1 to 6 Lyzo (g/l) IgA (g/l) Lysozyme LA (g/l) Month IgA IgM (g/l) Lactalbumin Month IgM LF (g/l) IgG (g/l) Lactoferrin Month IgG Month Month Month4 5 6 K Casein Abundance 2.00E E E E E+00 K Casein 0 2 Month b Casein Abundace 4.00E E E E E+00 b Casein 0 2 Month USA Brazil India South Africa Peru Bangladesh

39 Protein trends during lactation in countries Trends in proteins are consistent across countries.

40 Protein behavior in severely stunted children P = P = P = P = P = P =

41 Trends in IgG glycosylation in different countries Glycoforms change in the same pattern. IgG function and glycosylation. IgG glycosylation

42 Final Thoughts Get the best analytics possible Diet changes total abundance, but not relative abundances of HMO. HMO is everywhere. Both in the mother and in the infant. How does systemic HMO affect the mother? What role does systemic HMO have in infants? HMO important in stunting and in infant health. Protein/glycoproteins are yet unknown factors in infant development Glycoproteins do not all decrease during lactation. Complicated behavior and differs with protein. Glycoproteins function as pathogen block.

43 UC Davis Milk Bioactive Program Clinical conditions Autism Preterm birth Environmental enteropathy Infection Obesity Analytics Microbiome Compound isolation/pr oduction Immunity Host Defense/infectio n Gut Development Brain Development Microbiome Development

44 Bill and Melinda Gates Foundation