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

David Dallas

Assistant Professor Nutrition Program School of Biological and Population Health Sciences

Milk Protein Digestion in Premature Infants:

a Peptidomics and Enzyme Analysis Approach

www.dallaslab.org

Wesley, 2008

Milk proteins

Infant digestion

(Stevens, 2010, Epithelial Transport Physiology)

peptidase

Isolated milk protein

Anti- hypertensive

In vitro digestion

Calcium-binding Antimicrobial

Isolated enzyme

Immune modulation Opioid

Premature infant digestive system

• produce less gastric acid
• lower gastric pepsin and intestinal

protease activity than in term infants

• Lack of digestive capacity: critical
• Digestion of milk proteins = peptides with

antimicrobial and immunological activities

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.

Dry down and rehydrate supernatant C18 solid phase extraction Collect skim Inject 10-50 µL milk Precipitate protein lipid Centrifuge Folch

Collision gas Fragment ion Neutral loss Collision cell Precursor ions Activated ion Fragmenting ion Activated fragment ion (continues to fragment) Fragment ions (product ions)

Isolation, fragmentation and detection

Tandem spectra can be annotated manually.

QGGSSRA GGSSRA

• Q

GSSRA

• G

SSRA

• G

SRA 333.188

• S

AV 171.113 AVADTRDQADGSRASVDSGSSE 1081.981 AVADTRDQADGSRASVDSGSS 1017.460 AVADTRDQADGSRASVDSGS 973.944 AVADTRDQADGSRASVDSG 930.428 AVADTRDQADGSRASVD 858.401

AVADTRDQADGSRASVDSGSSEEQGGSSRA from polymeric immunoglobulin receptor

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

u-PA t-PA

Plasmin Plasminogen

Type 1 plasminogen activator inhibitor α-2 antiplasmin

X X

SERPINA5

X

Kallikrein

Kallistatin SERPING1

X

Thrombin Prothrombin

Antithrombin III Thrombin inhibitor

X

Elastase Proelastase Trypsinogen Trypsin

X

Prekallikrein Procathepsin D Cathepsin D

α-1-antichymotrypsin

X X

Inter-α-trypsin inhibitor α-1-antitrypsin Anti-elastase

X X

Dallas et al. (2015)

Bioinformatic Approach

• Map cleavage sites
• Compare to enzyme

specificity tables Active Enzymes:

• plasmin
• elastase
• cathepsin D
• carboxypeptidase B

(Khaldi, Dallas et al., JAFC)

human milk or preterm gastric samples (2X) A) Add supernatant samples to tube Add standards and blanks in other tubes B) Add buffer and synthetic substrate* and incubate at 37°C for 60 min Centrifuge at 3,000 rpm, 10 min at 4°C C) Transfer in a microplate D) Read with a microplate reader

Fluorophore

• Total protease
• Plasmin
• Elastase
• Kallikrein
• Thrombin
• Cathepsin D
• Carboxypeptidase

Activity was determined for:

y = 2568.5x - 2287 R² = 0.99914 50000 100000 150000 200000 250000 300000 20 40 60 80 100

Value fluorescence RFU (485/535 nm) Standard protease (ng/mL)

Protease activity by fluorometric or spectrometric assays

Many proteases not only present, but active in human milk!

• Identified proteases
• By abundance (high to low)

– Carboxypeptidase B2 – Plasmin – Kallikrein – Elastase – Thrombin – Cathepsin D – Cytosol aminopeptidase

• By activity (high to low)

– Kallikrein – Carboxypeptidase – Cathepsin D – Plasmin – Thrombin – Elastase – Cytosol aminopeptidase

Demers-Mathieu et al., submitted to Journal of Nutrition Veronique 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?

Common name Number of peptides β–casein 316 Polymeric immunoglobulin receptor 54 Osteopontin 41 Butyrophilin 27 α s1-casein 25 Mucin 1 9 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)

Results

Not digested:

• Lactoferrin
• α-lactalbumin
• Immunoglobulins

Peptide release is selective!

What functions do the peptides have?

Building a functional milk peptide database

Functional milk peptide database

Initiated at UC Davis (200 peptides) 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

• Many ways to search (identical,

truncated, precursor and homology)

• Batch search (multiple

sequences)

• Many additional search options

(e.g. function, category, species, protein).

Online tool for peptide functional searching

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)

50 100 150 200 250 300 350 400 450

<14 14-28 29-41 42-58 Number of peptides

Lactation period

Preterm Term

*** *** **

Peptides identified by protein

• CASB = β-casein
• OSTP = osteopontin
• CASA1 = αs1-casein
• PIGR = polymeric immunoglobulin receptor

20 40 60 80 100 120 140 160 180 200

CASB OSTP CASA1 PIGR Other

Number of peptides

Preterm Term

*** *** ** *** ***

Enzyme Activity

0.0E+00 4.0E+07 8.0E+07 1.2E+08 1.6E+08 2.0E+08

Plasmin/trypsin Carboxypeptidase B2 Cytosol aminopeptidase Cathepsin D Elastase

Enzyme activity (ion counts)

Enzyme

Preterm Term * ***

4.0x107 1.2x108 8.0x107 1.6x108 2.0x108 0.0

Specificity of enzymes in protein digestion

0.00E+00 5.00E+06 1.00E+07 1.50E+07 2.00E+07 2.50E+07

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

Abundance (ion counts)

αs1-casein amino acid position Preterm Standard error: preterm Term Standard error: term

N-terminus 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

Digestion is occurring despite high pH and low predicted pepsin activity

50 100 150 200 250 300 350 400 450 500 milk gastric Number of peptides found P-value: 1.29E-06

50 100 150 200 250

Number of peptides

10 20 30 40 50 60

Protein

milk gastric ** ** * ** ** ** ** * (Dallas et al., 2014, JN, accepted)

Results

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 k Gastric Re f

αs1-CN FFVAPFPEVFGK Bovine ACE-inhibitory x x αs1-CN IGSENSEKTTMP Bovine ACE-inhibitory x αs1-CN LRLKKYKVPQL Bovine Antimicrobial x x 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 αs1-CN YLGYLEQLLR Bovine Anxiolytic x x αs2-CN ALNEINQFYQK Bovine ACE-inhibitory x αs2-CN ALPQYLKTVYQHQKAMKPWIQPKTKVIPYV RYL Bovine Antimicrobial x 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 enteritis x β-CN HKEMPFPK Bovine Antimicrobial x x 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 β-CN RELEELNVPGEIVESLSSSEESITR Bovine CPP x β-CN VENLHLPLPLL Bovine ACE-inhibitory x β-CN VKEAMAPK Bovine Antioxidant x x β-CN VLPVPQKAVPYPQR Bovine Antimicrobial x β-CN WSVPQPK Human Antioxidant x x β-CN YQEPVLGPVR Bovine ACE-inhibitory x 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

Using the similarity search function with a 90% similarity threshold: bioactive peptides identified in the gastric of infants

277

100% matches

Are bioactive peptides released?

Which enzymes are active in the infant stomach?

Many proteases differed in activity in the stomach

• pH decreased from milk (6.35) to gastric (4.67)
• Cathepsin D, elastase increased
• Kallikrein, cytosol aminopeptidase decreased
• Pepsin detected in stomach, not milk
• Estimated that at least 90.2% of total

proteolytic activity in stomach derives from milk proteases rather than pepsin

2 4 6 8 10 12 Milk Gastric Elastase activity (ng/mL)

***

250 500 750 1,000 1,250 1,500 Milk Gastric Cathepsin D activity (ng/mL)

***

5 10 15 20 25 Milk Gastric Kallikrein activity (µg/mL)

**

1 2 3 4 Milk Gastric Pepsin concentration (ng/mL)

***

N.D.

pH optima: 8.0 pH optima: 4.0 pH optima: 6.5 pH optima: 2.0

5 10 15 20 25 30 35 EDOL LDOL EDOL LDOL EGA LGA Elastase activity (ng/mL)

***

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

Melinda Spooner Andi Markell, RD, LD

Robert Huston, MD

Randall Children’s Hospital, Legacy Emanuel, Portland, OR

• 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

Brian Scottoline, MD, PhD

Doernbecher Children’s Hospital, Oregon Health and Sciences University, Portland, OR

How do common breast milk handling practices affect the milk proteases and peptides?

Samples aliquots

Dry ice

Refrigeration 4ºC 1-6 days

Room temperature (1-6 hrs.)

Water bath thaw 37ºC Refrigerator thaw 4ºC

OR

• 80ºC

Freezer until analysis

Analysis

y = 223.04x + 101.43 R² = 0.99034 100 200 300 400 500 600 700 800 900 0.5 1 1.5 2 2.5 3 3.5

absorbance (nm) Concentration (µL)

ELISA and Enzyme substrate assays

• Total enzyme activity
• Each milk protease

Peptidomic Analysis

• Quantifies peptides

released

SDS-PAGE Gel

• Protein profile

Donor milk project

• How do different treatments affect milk proteases, milk proteins

and peptides?

– Batch pasteurization

• HTST

– Sterilization

• others (UV-C)

Carly Robertson Casey Collins Ashley Victor

Honglip Park Jason Foss Kimber Kirschner Zoha Ahmad Nicole McGuire Kelly Hollenbeck

Oregon State University Post-docs Soeren Drud Nielsen Veronique Demers- Mathieu Grad students Melinda Spooner Robert Beverly Research assistant Yuki Qu Interns Casey Collins Carly Robertson Ashley Victor Honglip Park Jason Foss Kimber Kirschner Zoha Ahmad Nicole McGuire Kelly Hollenbeck UC Davis Collaborators/mento rs

• Dr. J. Bruce German
• Dr. Carlito Lebrilla
• Dr. Andres

Guerrero

• Evan Parker
• Dr. Mark Underwood
• Dr. Nora Khaldi

Grad students Randall Robinson Tian Tian

Collaborators

Acknowledgements

Current Funding

• NIH NICHD K99/R00 Career Award

dave.dallas@oregonstate.edu

www.dallaslab.org

Current Funding