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) Enzymeactivity (ion

0.0E+000.0 Plasmin/trypsin Carboxypeptidase Cytosol Cathepsin D Elastase B2 aminopeptidase Enzyme Specificity of enzymes in protein digestion

2.50E+07 Preterm Standard error: preterm 2.00E+07 Term Standard error: term

1.50E+07

1.00E+07

5.00E+06 Abundance (ion counts) (ion Abundance

0.00E+00 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 111 116 101 106 121 126 131 136 141 146 151 156 161 166 171 176 181

N-terminus C-terminus αs1-casein amino acid position 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 the β-CN WSVPQPK Human Antioxidant x x β-CN YQEPVLGPVR Bovine ACE-inhibitory x gastric of infants β-CN YQEPVLGPVRG Bovine ACE-inhibitory x β-CN YQEPVLGPVRGPFPI Bovine Antimicrobial x x β-CN YQEPVLGPVRGPFPIIV Bovine immunomodulatory x x β-LG DAQSAPLRVY Bovine ACE-inhibitory x β-LG IDALNENK Bovine stimulates proliferation x x β-LG IIAEKTKIPAVF Bovine Antimicrobial x β-LG LDAQSAPLR Bovine ACE-inhibitory x β-LG LDIQKVAGTW Bovine ACE-inhibitory x β-LG LIVTQTMK Bovine Cytotoxic x x β-LG TPEVDDEALEK Bovine DPP-IV Inhibitor x κ-CN HPHPHLSF Bovine ACE-inhibitory x x κ-CN MAIPPKKNQDKTEIPTINT Bovine Antimicrobial x Which enzymes are active in the infant stomach? Many proteases differed in activity in the stomach 12 1,500 25 10 ** 4 *** 1,250 *** 20 ***

8 1,000 ) 15 3 6 750 g/mL (ng/mL) µ ( (ng/mL) 10 4 500 2 Elastaseactivity (ng/mL) 2 250 Kallikreinactivity 5 CathepsinDactivity 1 0 0 0 Milk Gastric Pepsinconcentration N.D. Milk Gastric Milk Gastric 0 pH optima: 8.0 pH optima: 4.0 pH optima: 6.5 Milk Gastric pH optima: 2.0 • pH decreased from milk (6.35) to gastric (4.67) 35 30 *** • Cathepsin D, elastase increased 25 • 20 Kallikrein, cytosol aminopeptidase decreased 15 • Pepsin detected in stomach, not milk 10 5 • Estimated that at least 90.2% of total

Elastaseactivity (ng/mL) 0 EDOL LDOL EDOL LDOL proteolytic activity in stomach derives from EGA LGA milk proteases rather than pepsin Preterm vs. term mother’s milk and infant gastric digestion • How does digestion differ across gestational age? – Compare early preterm, late preterm and term – Measure activity and concentration of proteases (in progress) – Measure digestion of milk proteins by peptidomics (in progress) Summary

• Digestion is occurring in the stomach • Most cleavage due to milk proteases • Digestion similar for human and bovine proteins • Hundreds of peptides homologous with functional peptides • Proteases more active in term than preterm stomach New infant study • Infants across gestational age and weight at birth, tracked throughout hospital stay • Milk, gastric, intestinal, stool, urine collection • Complete protein picture: amino acid analysis, peptidomics and protein profiling Doernbecher Children’s Hospital, Randall Children’s Hospital, Oregon Health and Sciences Legacy Emanuel, Portland, OR University, Portland, OR

Brian Scottoline, MD, PhD Melinda Spooner Andi Markell, RD, LD Robert Huston, MD How do common breast milk handling practices affect the milk proteases and peptides? Dry ice

Refrigeration 4ºC 1-6 days

Samples aliquots

Room temperature -80ºC (1-6 hrs.) Freezer until OR analysis Refrigerator Water bath thaw 4ºC thaw 37ºC Analysis

900

800 y = 223.04x + 101.43 R² = 0.99034 700

600

500

400 absorbance (nm) 300

200

100

0 0 0.5 1 1.5 2 2.5 3 3.5 Concentration (µL) ELISA and Enzyme Peptidomic Analysis SDS-PAGE Gel substrate assays • Quantifies peptides • Protein profile • Total enzyme activity released • Each milk protease Donor milk project • How do different treatments affect milk proteases, milk proteins and peptides? – Batch pasteurization -HTST – Sterilization -others (UV-C)

Honglip Park Jason Foss Kimber Kirschner Zoha Ahmad Nicole McGuire Kelly Hollenbeck Carly Robertson Casey Collins Ashley Victor Acknowledgements

Collaborators Current Funding Oregon State Jason Foss UC Davis Current Funding University Kimber Kirschner Collaborators/mento • NIH NICHD K99/R00 Career Award Post-docs Zoha Ahmad rs Soeren Drud Nielsen Nicole McGuire Dr. J. Bruce German Veronique Demers- Kelly Hollenbeck Dr. Carlito Lebrilla Mathieu - Dr. Andres Grad students Guerrero Melinda Spooner - Evan Parker Robert Beverly Dr. Mark Underwood Research assistant Dr. Nora Khaldi Yuki Qu Grad students Interns Randall Robinson Casey Collins Tian Tian Carly Robertson Ashley Victor [email protected] Honglip Park www.dallaslab.org