Presentation

ENGINEERING OF DAIRY PROTEINS AND THE MODULATION OF 

THEIR STRUCTURES - 

MICELLISATION AND IMMUNO REACTIVITIES OF 

DIMERIC DIMERIC BETA BETA CASEINS CASEINS. 

Thomas Haertlé 

Institut National de la Recherche Agronomique, Nantes, France 

The France-Egypt Year Of Science And Technology, 2010

Importance of milk 

Essential food of newborn (nutrition, immune protection, 

enzymes). 

Cow, goat and sheep in Western countries. 

Mare, water buffalo, camel, yak…in y Eastern countries. 

Consumed without or after transformation (cheeses, fermented 

milks). 

Of great importance for food industries of each human society. 


Average composition 

of f cow milk ilk 

Synthetisised by secretory epithelial cells (synthesis of 

components or their capture from blood) 

12 12-13 13 % of fd dry matter tt (120-130 (120 130 g/L) /L) 

PProteins i (32-35 g/L) LLactose (47-52 g/L) FFats 

(33-45 g/L) 

Caseins (26-28 ( g/L) g ) 

Precipitation at pH 4,6 

The France-Egypt Year Of Science And Technology, 2010 

Salt min. 

(7 g/L) 

BLG 

(2 (2-44 g/L) /L) 

Wh Whey 

ALA 

(1-1,5 g/L) LF 

Ig BSA

Bovine caseins 

Casein αS1 αS2 β κ 

AA 

199 

207 

209 

169 

MM (kDa) 23,6 25,25 

24 

19 

Variants 5 

3 

6 

6 

Cys 

0 

2 

0 

2 

S-S Bridges 0 

0 

0 

0 

Ser-P Ser P 

8 

11 

5 

2 

Glycosylation No 

No 

No 

Yes 

Major ep. 6 

4 

6 

8 

Minor ep. 3 66 

3 0 

UUnknown k th three-dimensional di i l structures t t 

Unknown functions (nutritional, only ?) 

Quite often allergenic. 


Caséine κ 

169 aa 

19037 Da 

1 

Chymosine 

Glycosylation 

Cys y Cys y 

P of caseins 

11 Para-Kappa 88 105/106 CMP 149 

Hydrophobe 

Hydrophile/chargé - 

Caséine β 

209 aa 

23983 Da 

Amphiphile 

Plasmine Plasmine 

P PPP 

P 

1 15 17-19 35 

105/106 

209 

28/29 107/108 


Caséine αs2 207 aa 

25226 Da 

Amphiphile 

PPP P P CysCys PPP 

P 

P P P 

Tè Très peu chargé h é 

Hydrophobe 

1 8-10 16 31 56 56-58 5 61 

129 131 143 

207 

36 40 


Caséine αs1 199 aa 

23615 Da 

Hydrophobe 

Dipôle 

PP PPP P P P 

1 46 48 64-68 

75 115 

199 

Hydrophobe Hydrophile/chargé - Hydrophobe Hydrophobe 

169 


Physico-chemical properties 

of caseins 

Casein micellisation according to Holt 

(1998)

20 - 300 nm 

(Schmidt, Schmidt, 1982 ; Walstra, 1984) 

1984 

Casein micelle 


glucids 

submicelle 

α αs11 αs β 2 

κ 

CCP Colloidal Calcium 

Phosphate

P 

P P P 

β casein 

MKVLILACLV ALALARELEE LNVPGEIVES LSSSEESITR INKKIEKFQS 

EEQQQTEDEL QDKIHPFAQT QSLVYPFPGP IPNSLPQNIP PLTQTPVVVP 

PFLQPEVMGV Q SKVKEAMAPK HKEMPFPKYP VEPFTESQSL Q TLTDVENLHL 

PLPLLQSWMH QPHQPLPPTV MFPPQSVLSL SQSKVLPVPQ KAVPYPQRDM 

PIQAFLLYQE PVLGPVRGPF PIIV 

Phosphorylation calcium fixation (binding of other caseins by 

phosphocalcic bridges) 

Hydrophilic NN-terminus terminus (several negative charges) 

Long hydrophobe C-terminal sequence 

AMPHIPHILIC PROTEIN 

structures polyproline II 

Numerous Numerous prolines (35) scarce classic secondary structures (β sheets, sheets α helices …) 

) 


P

Self-association of β casein 

Micellar aggregates 

Temperature and 

concentration ddependent d 


Andrews et al. , 1979 

Payens et al., 1963 

Farrell et al., 2001 

Thurn et al. , 1987 

Kajiwara et al. , 1988 

Sood et al. , 1990 

Sood et al. , 1997 

Leclerc et al. , 1998 

Leclerc et al. , 1997

F I 

Trp 143 

monomer 

λ 

↑ °CC 

Intrinsic Fluorescence of 

tryptophan tryptophan (1) (1) 

F I 

micelle 

λ max Trp = 341 nm λ max Trp = 336 nm 


λ

Enginering of β casein 

Recombinant β casein, wild type, expressed in E. coli : WT 

Recombinant β casein, mutated, expressed in E. coli: MU 

MRELEEL RELEELNVPG EIVESLSSSE ESITRINKKI EKFQSEEQQQ 

TEDELQDKIH PFAQTQSLVY PFPGPIPNSL PQNIPPLTQT PVVVPPFLQP 

EVMGVSKVKE AMAPKHKEMP FPKYPVEPFT ESQSLTLTDV ENLHLPLPLL 

QSWMHQPHQP LPPTVMFPPQ SVLSLSQSKV LPVPQKAVPY PQRDMPIQAF 

LLYQEPVLGP VRGPFPIIV 

Sequence containing three glutamates well conserved in Ruminants 

Addition of three negative charges: change of hydrophilic/hydrophobic ratio 


Same conditions as 

for DLS. 

λmmax 

Trp 

1 mg/mL 

00,4 4 mg/mL 

λmaxTrp 

0,2mg/mL 

λmaxTrp max Trp 

λm 

342 

340 

338 

336 

334 

332 

342 

340 

338 

336 

334 

332 

Native 

Intrinsic Fluorescence of 

Tryptophan Tryptophan (1) (1) 

(2) 

10 20 30 40 50 

Temperature, °C 

WT 

10 20 30 40 50 

T emperature, t °C 

Temperature, °C 

λmmaxTrp 

max Trp 

λm 


342 

340 

338 

336 

334 

332 

MU 

10 20 30 40 50 

Temperature, °C

Beta casein native 

+ 3 HNeRELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNS 

3 

LPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQ 

SWMHQPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV 

COO - 

Beta casein simple mutant C4 

+ 3 HNeMGRELECLNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTP 

VVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSL 

SQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV COO - 

Q Q Q Q Q 

Beta casein simple mutant C208 

+ 3 HNeMGRELEKLNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPV 

VVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSLS 

QSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPICV COO - 

Beta casein double mutant C4 - 208 

+ 3 HNeMGRELECLNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDELQDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTP 

VVVPPFLQPEVMGVSKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLSL 

SQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPICV COO - 


SDS-PAGE of bovine β casein and mutants. 

Experimental condition: SDS‐PAGE (12.5 % gel), under reducing conditions. 

Coomasie blue staining. 


SDS-PAGE 

BCN C4- 

208 

Oxydation 

24 h at 25°C pH 6,5 

WT 

BCN C4 BCN C4- 

208 

BCN C4 WT 

kDa 0 1 10 24 M 0 1 10 24 0 1 10 24 M 0 1 10 24 

97,4 

97,4 

Trimer 

66,2 

Dimer 

Monomer 

«Intra» a 

66,2 

45 

31 

2 cys 1 cys 


2-mercaptoethanol 

kDa 

45 

31 

21,5

SDS-PAGE of the dimerizing β casein mutants 

Condition: Tris buffer 50 mM mM, pH 88.2 2 including 80 mM NaCl and H2O2 H2O2, 37 °CC, 

stirred, sterile. 


• RReasons ffor quicker i k dimerization di i ti kinetics ki ti of f C208 ßß-CNs CN 

Greater mobility y of C-terminal segment g 

Easier access of SH function 

Hydrophobic interactions 

• The dimerization sites in C6- and C-208 ß-CNs 

• RELECSH(4)NVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDEL 

QDKIHPFAQTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGV 

• SKVKEAMAPKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMH 

• QPHQPLPPTVMFPPQSVLSLSQSKVLPVPQKAVPYPQRDM PIQAFLLYQ 

• EPVLGPVRGPF PIIVCSH (208) 


Hydrophilic Hydrophilic-Hydrophilic Hydrophilic patterns of different β CNs 

Native β CN has distinct arrangement of hydrophilic (ψ) -hydrophobic(φ) 

fragments (ψ- φ). 

The dimeric β CNs are in some sense symmetrical 

C208 β CND (ψ - φ ~ φ - ψ) 

C4 β CND (φ - ψ ~ ψ - φ) 


"P "Palindromic" lid i"

Casein β 

-C-C- 

-C-C- C C 

23983 Da 

Plasmin Plasmin 

P PPP 

P 

1 15 17 17-19 19 35 

105/106 

209 

28/29 107/108 

Hydrophilic/charged - 

Amphiphilic 

Little charged 

Hydrophobe 


Chhaperone-like 

activity ac (%) 

Chaperone-like activities of native and mutant monomeric 

ββ-CN. CN TTarget t protein t i - iinsulin li 

120 

100 

80 

60 

40 

20 

0 

0 0,025 0,05 0,075 0,1 0,125 

(ß-CN /Insulin) molar ratio 

Native β-CN (■), Wild type β-CN (□), C4 β-CN (▲) and C208 β-CN (∆) 


Chemically-induced 

aggregation of insulin 

performed f di in the th présence é of f 

20 mM DTT at 40 °C. 

Mutant β-CNs exhibit 

considerably smaller 

chaperone chaperone-activities activities than that 

of native β-CN.

Importance of phosphoryl residues for chaperone 

acivities of ßß-CNs. CNs 

The hydrophilic segment of chaperones plays an essential role 

increasing solubilities of target proteins. 

The expressed in E. coli recombinant ß-CNs are not phosphorylated 

what decreases their amphiphilicity 

amphiphilicity. 

Amino acid Sequence of native ß-CN and the sites for phosphorylation 

Smaller polarity of hydrophilic domain is the reason for poorer 

chaperone p 

activities of the mutant ß-CNs. 


Consequence of Cys incorporation on chaperone-like 

chaperone like 

activities of ß-CN. 

Absence of cysteinyl residues, is one of the common features among 

different chaperone families. 

The C4 ß-CN and C208 ß-CN have cysteinyl residue in position 4 and 208, 

respectively. 

The ß-CNs containing cysteine showed almost similar chaperone-like 

activities ti iti as WT ßß-CN. CN 

Thus incorporation of cysteine has no significant consequence in 

chaperone chaperone-like like acti activity it of monomeric ßß-CN. CN 


Chape erone-like activity y (%) 

120 

100 

80 

60 

40 

20 

0 

Chaperone-like activities of the dimeric β-CN. 

TTarget t protein-ADH. t i ADH 

0 0,25 0,5 0,75 1 1,25 1,5 1,75 

(β−CN/ADH) molar ratio 

C208 ββ-CND CND (▲) (▲): HHydrophobic d h bi core, Pl Polar ends. d 

C4 β-CND (∆): Polar core, Hydrophobic ends. 


Due to combination of flexibility and 

amphiphilicity, ß-CN acts as a surfactant 

molecule in solution. 

The dimeric β-CNs resembles 

somehow bis (gemini) - surfactants. 

C208 β-CND (ψ ‐ φ ~ φ ‐ ψ) has 

greater chaperone activity than C6 β- 

CND (ψ ‐ φ ~ φ ‐ ψ). 

C208 β-CND also shows greater 

chaperone activity than mutant β-CNs β CNs in 

monomeric state.

− 

Average IgE 

(ng/ml) 

35 

30 

25 

20 

15 

The IgE-binding (immuno reactivity) of different molecular 

forms of ββ-CN. CN 

Sera from patients with milk 

allergies were used to 

determine immuno reactivities 

of different molecular forms of 

β-CN. 

Averaged results of 16 out of 

10 37 tested sera showing 

significantly greater IgE- 

5 

responses to different forms 

of ββ-CN CN 

0 

Native B-CN WT B-CN C4 B-CNM C208 B-CNM C4 B-CND C208 B-CND 


Summary 

ß-CN and its mutants undergo temperature-induced concentrationdependent 

micellisation. 

Polar phosphate residues and the NN-terminal terminal hydrophilic domain are 

important functional elements enhancing the chaperone-like activities of 

native β-CN. 

Dimerization of C208 β-CN with two distal hydrophilic domains improved 

considerably its chaperone-like activity in comparison with its monomeric 

form and with C4 β-CND. 

NN-terminal t i l hydrophilic h d hili domain d i plays l significant i ifi t role l as important i t t 

functional elements enhancing the chaperone-like activity of native β-CN 

Exposure of hydrophobic domains increases immuno reactivity in 208 ββ-CN CN 

dimer 


Our INRA team 

Isabelle Bronnec Aynur Akhmadova – PhD student 

JJean-Marc M Ch Chobert b t Sh Shady d El GGaish i h – PhD student t d t 

Yvan Choiset Tatyana Konnova – PhD student 

Michèle Dalgalarrondo Asghar Taheri Kafrani – PhD student 

Hanitra Rabesona Iuliya Schutskaya – PhD student 

Thomas Haertlé Reza Yousefi – post doctoral 

Collaborations 

Prof Dr V.I. Muronetz, Faculty of Bioengineering, Lomonosov University, 

Moscow, Russia 

Prof Dr Yu. D. Zuev, Institute of Biochemistry and Biophysics, RAS, 

Kazan, Russia. 

Prof Dr A. A. Moosavi-Movahedi, Institute of Biochemistry and Biophysics, 

Tehran University, IR Iran.

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