Milk Protein Digestion in Premature Infants: a Peptidomics and Enzyme Analysis Approach David Dallas Assistant Professor Nutrition Program School of Biological and Population Health Sciences www.dallaslab.org Milk proteins Wesley, 2008 Infant digestion peptidase (Stevens, 2010, Epithelial Transport Physiology) Isolated Antimicrobial enzyme Anti- hypertensive In vitro digestion Calcium-binding Immune modulation Opioid Isolated milk protein Premature infant digestive system • produce less gastric acid • lower gastric pepsin and intestinal protease activity than in term infants Preterm Term Adult Pepsin activity1 (U/mL) 12 125 (10X) 600 (50X) Gastric pH 2 4.1 – 5.8 3.2 – 5.0 1.8 – 2.0 Elastase level3 (µg/g) 113 – 127 129 – 160 > 200 Adapted from Henderson et al. (2001)1, Armand et al. (1995, 1996)1, Mason (1962)2, Kori et al. (2016)3. • Lack of digestive capacity: critical • Digestion of milk proteins = peptides with antimicrobial and immunological activities Collect Folch Precipitate Centrifuge skim protein lipid 10-50 µL milk supernatant C18 solid phase extraction Dry down and Inject rehydrate Isolation, fragmentation and detection Collision Fragment Neutral Collision gas ion loss cell Precursor Fragment ions ions (product ions) Activated Fragmenting Activated fragment ion ion ion (continues to fragment) Tandem spectra can be annotated manually. AVADTRDQADGSRASVDSGSSEEQGGSSRA from polymeric immunoglobulin receptor GGSSRA AVADTRDQADGSRASVDSGSSE 1081.981 AVADTRDQADGSRASVDSGSS 1017.460 AVADTRDQADGSRASVDSGS 973.944 AVADTRDQADGSRASVDSG 930.428 QGGSSRA AVADTRDQADGSRASVD 858.401 SRA -Q AV 333.188 GSSRA 171.113 SSRA -G -G -S Does milk contain peptides? • Assumption: Only intact proteins • Findings: – Yes, approximately 300 peptides present • Our new research shows 1-2 thousand – Mostly the same peptides for all healthy mothers (Dallas et al., 2013. J. Proteome Research. “Extensive in vivo milk peptidomics reveals specific proteolysis yielding protective antimicrobial peptides”) Which enzymes cleave the proteins? Milk contains complex system of proteases and antiproteases Type 1 plasminogen activator inhibitor α-2 antiplasmin Prothrombin Plasminogen X u-PA t-PA Thrombin X Trypsinogen Plasmin X Antithrombin III Prekallikrein Thrombin inhibitor X Proelastase Trypsin Kallikrein Inter-α-trypsin inhibitor Elastase X α-1-antitrypsin X X SERPINA5 Kallistatin X SERPING1 Procathepsin D Anti-elastase X α-1-antichymotrypsin X Cathepsin D Dallas et al. (2015) Bioinformatic Approach Active Enzymes: • Map cleavage sites -plasmin • Compare to enzyme -elastase specificity tables -cathepsin D -carboxypeptidase B (Khaldi, Dallas et al., JAFC) Protease activity by fluorometric or spectrometric assays A) Add supernatant human milk or preterm samples to tube D) Read with a microplate reader gastric samples (2X) Add standards and blanks in other tubes B) Add buffer and synthetic substrate* and incubate at 37°C C) Transfer in a microplate Centrifuge at 3,000 for 60 min rpm, 10 min at 4°C Activity was determined for: 300000 • Total protease 250000 • Plasmin 200000 • Elastase 150000 • Kallikrein 100000 y = 2568.5x - 2287 R² = 0.99914 • Thrombin (485/535nm) 50000 0 • Cathepsin D Valuefluorescence RFU 0 20 40 60 80 100 Fluorophore • Carboxypeptidase Standard protease (ng/mL) Many proteases not only present, but active in human milk! • Identified proteases • By abundance (high to low) • By activity (high to low) – Carboxypeptidase B2 – Kallikrein – Plasmin – Carboxypeptidase – Kallikrein – Cathepsin D – Elastase – Plasmin – Thrombin – Thrombin – Cathepsin D – Elastase – Cytosol aminopeptidase – Cytosol aminopeptidase Veronique Demers-Mathieu et al., submitted to Journal of Nutrition Demers-Mathieu Human milk protease inhibitors found • By abundance (high to low) – α1-antitrypsin – Antithrombin III – α1-antichymotrypsin – α2-antiplasmin – plasma serine protease inhibitor Is mammary gland digestion protein-selective? Results Common name Number of Not digested: peptides β–casein 316 - Lactoferrin Polymeric immunoglobulin receptor 54 - α-lactalbumin Osteopontin 41 Butyrophilin 27 - Immunoglobulins α s1-casein 25 Mucin 1 9 Peptide release is selective! κ-casein 8 Perilpin-2 5 Bile salt-activated lipase 2 Lactoperoxidase 2 Macrophage mannose receptor 2 Misshapen-like kinase 1 2 Sialic acid binding Ig-like lectin 9 2 Proteins with only 1 unique peptide 13 (Dallas et al., 2013. J. Proteome Research) What functions do the peptides have? Building a functional milk peptide database Initiated at UC Davis (200 peptides) Functional milk peptide database Expanded and improved at OSU: Milk Bioactive Peptide Database MBPDB (892 peptides) • 2,801 articles mined -> 254 with unique original identifications • Carefully referenced • Publishing public database Soeren Drud Nielsen Rob Beverly Yuki Qu Online tool for peptide functional searching • Many ways to search (identical, truncated, precursor and homology) • Batch search (multiple sequences) • Many additional search options (e.g. function, category, species, protein). Functional milk peptide database • Antihypertensive • Antimicrobial • Antioxidant • Anti-inflammatory • Opioid • Etc. • Across all available milk species, includes bovine, human, goat, sheep, camel, pig, etc. Mapping function, frequency and activity Homology search Homology search Antimicrobial peptide Functional prediction Soeren Drud Nielsen Predicting functional peptides across the protein sequence Are peptides different between term and preterm milk over lactation? Peptide Count • Effect of lactation stage: not significant • Effect of maturation: significant <0.001) 450 Preterm *** *** Term 400 ** 350 300 250 200 150 100 50 0 Number of peptides <14 14-28 29-41 42-58 Lactation period Peptides identified by protein 200 *** Preterm Term 180 160 140 120 100 80 *** *** ** 60 *** 40 Number of peptides 20 0 CASB OSTP CASA1 PIGR Other • CASB = β-casein • OSTP = osteopontin • CASA1 = αs1-casein • PIGR = polymeric immunoglobulin receptor Enzyme Activity 2.0x108 2.0E+08 Preterm * Term 8 1.6x101.6E+08 1.2x101.2E+088 8.0x108.0E+077 *** 4.0x104.0E+077 Enzyme activity (ion counts) Enzyme activity(ion 0.0E+000.0 Plasmin/trypsin Carboxypeptidase Cytosol Cathepsin D Elastase B2 aminopeptidase Enzyme Specificity of enzymes in protein digestion Abundance (ion counts) 1.50E+07 2.00E+07 2.50E+07 0.00E+00 5.00E+06 1.00E+07 N - terminus 1 6 11 16 21 26 α 31 s1 36 - 41 casein 46 51 56 61 66 amino 71 76 81 86 91 acid 96 101 106 Standard error:term Term Standard error:preterm Preterm position 111 116 121 126 131 136 141 146 151 C 156 - terminus 161 166 171 176 181 Summary • Greater abundance of peptides and enzyme activity in pre-term milk • Preterm infants are receiving substantially different milk! Among mother’s of preterm infants, do their milk proteases differ with length of gestation or infant day of life? Few differences in proteases in preterm mother’s milk across gestational age at delivery and time post partum • Groups – Early gestational age (24-26 weeks) – Late gestational age (27-32) • Mostly stayed constant! How much digestion occurs in the infant? What peptides are released? Results intact gastric (Dallas et al., 2014, JN, accepted. “A peptidomic analysis of human milk digestion in the infant stomach reveals protein-specific degradation patterns”) Results: peptides increase over 2-fold from intact to gastric sample 500 P-value: 1.29E-06 450 400 350 300 250 200 150 Number of peptides found 100 50 0 milk gastric Digestion is occurring despite high pH and low predicted pepsin activity Results 250 ** 60 50 ** 200 40 150 milk 30 100 gastric 20 ** ** 50 10 Number of peptides ** * ** * 0 0 Protein (Dallas et al., 2014, JN, accepted) Do preterm infants digest bovine and human milk proteins differently? • Preterm infants fed human milk enriched with human milk fortifier based on cow milk. • Highly similar amount of peptides released Human milk proteins Bovine milk proteins Protein sequence Species Function Mil Gastric Re k f αs1-CN FFVAPFPEVFGK Bovine ACE-inhibitory x x 100% matches αs1-CN IGSENSEKTTMP Bovine ACE-inhibitory x αs1-CN LRLKKYKVPQL Bovine Antimicrobial x x Are bioactive peptides αs1-CN RPKHPIKHQ Bovine ACE-inhibitory x x αs1-CN RPKHPIKHQGLPQEVLNENLLRF Bovine Antimicrobial x αs1-CN SDIPNPIGSENSEK Bovine Antimicrobial x x αs1-CN VLNENLLR Bovine Antimicrobial x released? αs1-CN YLGYLEQLLR Bovine Anxiolytic x x αs2-CN ALNEINQFYQK Bovine ACE-inhibitory x αs2-CN ALPQYLKTVYQHQKAMKPWIQPKTKVIPYV Bovine Antimicrobial x RYL αs2-CN AMKPWIQPK Bovine ACE-inhibitory x x αs2-CN FALPQYLK Bovine ACE-inhibitory x αs2-CN KTVYQHQKAMKPWIQPKTKVIPYVRYL Bovine Antimicrobial x αs2-CN LKKISQRYQKFALPQY Bovine Antimicrobial x αs2-CN TKVIPYVRYL Bovine Antimicrobial x αs2-CN VYQHQKAMKPWIQPKTKVIPYVRYL Bovine Antimicrobial x αs2-CN VYQHQKAMKPWIQPKTKVIPYVRYL Bovine Antimicrobial x αs2-CN YQKFPQY Bovine Antioxidant x x αs2-CN LKTVYQHQKAMKPWIQPKTKVIPYVRYL Bovine Antimicrobial x β-CN ENLHLPLPLL Human ACE-inhibitory x β-CN EPVLGPVRGPFP Bovine ACE-inhibitory x β-CN GVSKVKEAMAPKHKEMPFPKYPVEPFTESQ Bovine protective effects in x Using the similarity search function with a 90% enteritis β-CN HKEMPFPK Bovine Antimicrobial x x similarity threshold: β-CN LENLHLPLP Human ACE-inhibitory x β-CN MPFPKYPVEP Bovine ACE-inhibitory x β-CN PVVVPPFLQPE Bovine Antimicrobial x β-CN QEPVLGPVRGPFPIIV Bovine ACE-inhibitory x 277 β-CN RELEELNVPGEIVESLSSSEESITR Bovine CPP x β-CN VENLHLPLPLL Bovine ACE-inhibitory x bioactive peptides β-CN VKEAMAPK Bovine Antioxidant x x β-CN VLPVPQKAVPYPQR Bovine Antimicrobial x identified in