Milk Protein Digestion in Premature Infants: a Peptidomics and - PowerPoint PPT Presentation

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)

Antimicrobial Isolated enzyme Anti- hypertensive In vitro Calcium-binding digestion 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 activity 1 (U/mL) 12 125 (10X) 600 (50X) Gastric pH 2 4.1 – 5.8 3.2 – 5.0 1.8 – 2.0 Elastase level 3 ( µ g/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 Precipitate Folch skim Centrifuge protein lipid 10-50 µL milk supernatant C18 solid phase extraction Dry down and Inject rehydrate

Isolation, fragmentation and detection Collision Collision Fragment Neutral gas ion loss cell Fragment ions Precursor (product ions) 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 GSSRA AV 333.188 171.113 -G SSRA -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 Thrombin t-PA X Trypsinogen X Plasmin Antithrombin III Prekallikrein Thrombin inhibitor X Proelastase Trypsin Kallikrein Inter-α-trypsin inhibitor X Elastase X α-1-antitrypsin X SERPINA5 Kallistatin X SERPING1 Procathepsin D Anti-elastase X X Cathepsin D α-1-antichymotrypsin 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: Total protease • 300000 Plasmin • Value fluorescence RFU 250000 Elastase • 200000 (485/535 nm) 150000 • Kallikrein 100000 y = 2568.5x - 2287 Thrombin • R² = 0.99914 50000 Cathepsin D • 0 Carboxypeptidase • 0 20 40 60 80 100 Fluorophore Standard protease (ng/mL)

Many proteases not only present, but active in human milk! • Identified proteases • By activity (high to low) • By abundance (high to low) – Kallikrein – Carboxypeptidase B2 – Carboxypeptidase – Plasmin – Cathepsin D – Kallikrein – Plasmin – Elastase – Thrombin – Thrombin – Elastase – Cathepsin D – Cytosol aminopeptidase – Cytosol aminopeptidase Veronique Demers-Mathieu Demers-Mathieu et al., submitted to Journal of Nutrition

Human milk protease inhibitors found • By abundance (high to low) – α 1 -antitrypsin – Antithrombin III – α 1 -antichymotrypsin – α 2 -antiplasmin – plasma serine protease inhibitor

Is mammary gland digestion protein-selective?

Results Common name Number of Not digested: peptides - Lactoferrin β–casein 316 - α-lactalbumin Polymeric immunoglobulin receptor 54 Osteopontin 41 - Immunoglobulins Butyrophilin 27 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 Rob Beverly Yuki Qu Soeren Drud Nielsen

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) • Preterm 450 Term *** *** 400 ** 350 Number of peptides 300 250 200 150 100 50 0 <14 14-28 29-41 42-58 Lactation period

Peptides identified by protein *** 200 Preterm Term 180 Number of peptides 160 140 120 100 *** 80 *** ** 60 *** 40 20 0 CASB OSTP CASA1 PIGR Other CASB = β-casein • OSTP = osteopontin • • CASA1 = αs1-casein PIGR = polymeric immunoglobulin receptor •

Enzyme Activity 2.0x10 8 Preterm 2.0E+08 Enzyme activity (ion counts) Term * 1.6x10 8 1.6E+08 1.2E+08 1.2x10 8 8.0x10 7 8.0E+07 *** 4.0x10 7 4.0E+07 0.0E+00 0.0 Plasmin/trypsin Carboxypeptidase Cytosol Cathepsin D Elastase B2 aminopeptidase Enzyme

Specificity of enzymes in protein digestion Preterm 2.50E+07 Standard error: preterm Abundance (ion counts) Term 2.00E+07 Standard error: term 1.50E+07 1.00E+07 5.00E+06 0.00E+00 1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96 101 106 111 116 121 126 131 136 141 146 151 156 161 166 171 176 181 N-terminus α s1 -casein amino acid position C-terminus

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 Number of peptides found 350 300 250 200 150 100 50 0 milk gastric Digestion is occurring despite high pH and low predicted pepsin activity

Results ** 250 60 ** Number of peptides 50 200 40 milk 150 30 100 gastric 20 ** ** ** 50 * 10 ** * 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