T: +27(0)51 401 9111 | info@ufs.ac.za | www.ufs.ac.za

Casein and casein micelle structure

Prof G Osthoff

Contents Introduction : milk

Milk of other dairy animals: technological problems

Milk of other dairy animals: composition

Casein composition: interspecies comparison

Casein: structure and function

Casein micelle: structure

Casein micelle: interspecies comparison; answers?

Future

Acknowledgements

• Milk contains many nutrients:– lactose, proteins, fat, minerals

• Each of the nutrients in amounts much higher than is soluble.– How possible?

• Exist as suspension of insoluble colloidal forms– lipid vesicle and micelles

• Contains all enzymes for self destruction within minutes.– Why not??

• Enzymes as inactive forms or stored hidden from substrate

Milk

• Transfer of technology to milk of other species – problems!– Fouling heating systems– Sediment formation– Off flavours– Early gelation

• Reasons– Lower colloidal stability– Lower heat stability– Higher somatic cell counts– Stronger enzyme systems– Different protein composition

Other dairy animals: Technology problems

Species Alcohol stability(%)

Fat globule(µm)

Casein micelle(nm)

α-S1 casein (wt %)

α-S2 casein(wt %)

β-casein(wt %)

κ-casein(wt %)

Cow 75-85 3-4.5 100-140 38 10 40 12

Buffalo 60-69 4-7.5 200-260 40 9 35 12

Goat 60 2.5-3.5 50-200 20 16 41 17

Camel 65 2-3.9 260-300 65 22 9 4

Differences between dairy species

(Park, 2006)

OTHER DIFFERENCES Total Protein content Casein:Whey ratio Absence of β-lactoglobulin

CASEINS

Caseins

Family of phosphoproteins synthesised in the mammary gland in response to lactogenic hormones

Evolved from members of Secreted Calcium (Phosphate) binding Proteins (bone & tooth gene)

Eutherian milks: at least 3 and normally 4 typesαS1-, αS2-, β-, and κ-CN

Exist as colloidal aggregates - casein micelles Non-globular structure – No cysteine residues Cannot be crystallized Amphipathic nature Most structural knowledge- extrapolation from other proteins

(Horne, 2011 ; Mather, 2011; Holt, 2014 )

αs1- Casein

Major casein fraction of bovine milk

Highly phosphorylated → 2 forms (Serines)

Several variants exist (A, B & C forms)

Structure random, coils due to high amounts of Q and P. Helixes possible

Bind water(Model predicted structure)

Human MRLLILTCLVAVALARPKLPLRYPERLQNPSE------SSEPIPLESREEYMNGMNRQRN Cow MKLLILTCLVAVALARPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKVNEL----- Goat MKLLILTCLVAVALARPKHPINHQGLSPEVPNENLLRFVVAPFPEVFRKENINEL----- *:**************** *:.: : : *:* :* :* : Human ILREKQTDEIKDTRNESTQNCVVAEPEKMESSISSSSEEMSL------------------ Cow ---------SKDIGSESTEDQAMEDIKQMEAESISSSEEIVPNSVEQKHIQKDDVPSERY goat ---------SKDIGSESTEDQAMEDAKQMKAGSSSSSEEIVPNSAEQKYIQKEDVPSERY ** .***:: .: : ::*:: *****: Human SKCAEQFCRLNEYNQLQLQ--AAHAQEQIRRMNENSH-----------------VQVPFQ Cow LGYLEQLLRLKKYKVPQLEIVPNSAEERLHSMKEGIHAQQKEPMIGVNQELAYFYPELFR goat LGYLEQLLKLKKYNVPQLEIVPKSAEEQLHSMKEGNPAHQKQPMIAVNQELAYFYPQLFR **: :*::*: **: *:*::: *:* *: Human QLNQLAAYPYAVWYYPQ-IMQYVPFPPFSDISNPTAHENYEKNNVMLQW Cow QFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSGKTT-MPLW Goat QFYQLDAYPSGAWYYLPLGTQYTDAPSFSDIPNPIGSENSGKAA-MPLW *: ** *** ..*** **. * **** ** . ** * * *

(Ginger & Grigor, 1999; Horne, 2011; Mather, 2011; http://www.ebi.ac.uk/clustaO; Madende, 2016)

αs2- Casein

Highly and variably phosphorylated

• 4 differentially phosphorylated isoforms

Least hydrophobic Absent in human milk

(pseudogene present) Structure random, coils due to

high amounts of Q and P Helixes possible

Bind water(Model predicted structure)

(Ginger & Grigor, 1999; Horne, 2011; Mather, 2011; http://www.ebi.ac.uk/clustaO) ; Madende, 2016

Pig MKFFIFTCLLAVAFAKHEMEHVSSSEESINISQEKYKQEKNVINHPSKEDICATSCEEAV Cow MKFFIFTCLLAVALAKNTMEHVSSSEESI-ISQETYKQEKNMAINPSKENLCSTFCKEVV Goat MKFFIFTCLLAVALAKHKMEHVSSSEEPINIFQEIYKQEKNMAIHPRKEKLCTTSCEEVV *************:**. ********* * * ** ******: .* **.:*:* *:*.* Pig RNIKEVGYASSSSSEESVDIPAENVKVTVEDKHYLKQLEKISQFYQKFPQYLQALYQAQI Cow RNANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPI Goat RNANEEEYSIRSSSEESAEVAPEEIKITVDDKHYQKALNEINQFYQKFPQYLQYPYQGPI ** :* *: ******.:: *::*:**:**** * *::*.*********** **. * Pig VMNPWDQTKTSAYPFIPTVIQSGEELSTSEEPVSSSQEENTKTVDMESMEEFTKKTELTE Cow VLNPWDQVKRNAVPITPTLNR---------EQLSTSEENSKKTVDMESTEVFTKKTKLTE Goat VLNPWDQVKRNAGPFTPTVNR---------EQLSTSEENSKKTIDMESTEVFTKKTKLTE *:*****.* .* *: **: : * :*:*:*:..**:**** * *****:*** Pig EEKNRIKFLNKIKQYYQKFTWPQYIKTVHQKQKAMKPWNHIKTNSYQIIPNLRYF Cow EEKNRLNFLKKISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKV---IPYVRYL Goat EEKNRLNFLKIISQYYQKFAWPQYLKTVDQHQKAMKRWTQPKTNA---IPYVRYL *****::**: *.* ****: ***:*** *:***** * : **: ** :**:

β-Casein

Most hydrophobic casein Characteristic high proline Possess a very acidic N-

terminal sequence (Glu) Hydrolysis by plasmin yields

γ-casein One phosphorylated form and

3 variants in Bovine Structure random, coils due

to high amounts of Q and P Helixes possible

Bind water(Model predicted structure)

(Ginger & Grigor, 1999; Horne, 2011; Mather, 2011; http://www.ebi.ac.uk/clustaO) ; Madende, 2016

Human MKVLILACLVALALARE---------TIESLSSSEESITEYKQKVEKVKHEDQQQGEDEH Cow MKVLILACLVALALARELEELNVPGEIVESLSSSEESITRINKKIEKFQSEEQQQTEDEL Goat MKVLILACLVALAIAREQEELNVVGETVESLSSSEESITHINKKIEKFQSEEQQQTEDEL *************:*** :***********. ::*:**.: *:*** *** Human QDKIYPSFQPQPLIYPFVEPIPYGFLPQNILPLAQP-AVVLPVPQPEIMEVPKAKDTVYT Cow QDKIHPFAQTQSLVYPFPGPIHNS-LPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMAP Goat QDKIHPFAQAQSLVYPFTGPIPNS-LPQNILPLTQTPVVVPPFLQPEIMGVPKVKETMVP ****:* * * *:*** ** . ***** **:* .** *. ***:* * *.*::: Human KGRVMPVLKSPTIPFFDPQIPKLTDLENLHLPLPLLQPLMQQVPQPIPQTLALPPQPLWS Cow KHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPPTVMFPPQSVLS Goat KHKEMPFPKYPVEPFTESQSLTLTDVEKLHLPLPLVQSWMHQPPQPLSPTVMFPPQSVLS * : **. * *. ** : * .***:*:*******:* *:* **: *: :*** : * Human VPQPKVLPIPQQVVPYPQRAVPVQALLLNQELLLNPTHQIYPVTQPLAPVHNPISV Cow LSQSKVLPVPQKAVPYPQRDMPIQAFLLYQEPVLGPVRGPFPIIV----------- Goat LSQPKVLPVPQKAV--PQRDMPIQAFLLYQEPVLGPVRGPFPILV----------- : * ****:**:.* *** :*:**:** ** :* *.: :*:

κ-Casein Most studied casein (involved in

milk clotting) Insensitive to Ca2+ precipitation Only casein that is glycosylated

(Thr and Ser) N-terminal (+ve), C-terminal (-ve) Target for chymosin (Phe-

Met/Ile/Leu) 2 variants of bovine κ- CN (A &B) Structure random, coils due to high

amounts of P, Q and Y on N-term P, S and T rich C-term

(Model predicted structure)

Human -MKSFLLVVNALALTLPFLAVEVQNQKQPACHENDERPFYQKTAPYVPMYYVPNSYPYYG Cow MMKSFFLVVTILALTLPFLGAQEQNQEQPIRCEKDERFFSDKIAKYIPIQYVLSRYPSYG Goat MMKSFFLVVTILALTLPFLGAQEQNQEQPICCEKDERFFDDKIAKYIPIQYVLSRYPSYG ****:***. ********..: ***:** *:*** * :* * *:*: ** . ** ** Human TNLYQRRPAIAINNPYVPRTYYANPAVVRPHAQIPQRQYLPNSH--------PPTVVRRP Cow LNYYQQKPVALINNQFLPYPYYAKPAAVRSPAQILQWQVLSNTVPAKSCQAQPTTMARHP Goat LNYYQQRPVALINNQFLPYPYYAKPVAVRSPAQTLQWQVLPNTVPAKSCQDQPTTLARHP * **::*. *** ::* ***:*..** ** * * * *: * *:.*:* Human NLHPSFIAIPPKKIQDKIIIPTINTIATVEPTP--TPATEPTVDSVVTPEAFSESIITST Cow HPHLSFMAIPPKKNQDKTEIPTINTIASGEPTS--TPTTEAVESTVATLEDSPEVI-ESP Goat HPHLSFMAIPPKKDQDKTEVPAINTIASAEPTVHSTPTTEAIVNTVDNPEASSESI-ASA . * **:****** *** :*:*****: *** **:** .:* . * * * * Human PETTTVAVTPPTA Cow PEINTVQVTSTAV Goat SETNTAQVTSTEV * .*. ** .

(Ginger & Grigor, 1999; Horne, 2011; Mather, 2011; http://www.ebi.ac.uk/clustaO); Madende, 2016

CASEIN MICELLES & MODELS

Casein micelle models

(Horne, 1998)

A: a submicelleB: protruding chainC: Calcium phosphateD: κ-caseinE: phosphate groups

Colloidal calcium phosphate

Will crystallize in milk

Calcium binding in caseinsPig MKFFIFTCLLAVAFAKHEMEHVSSSEESINISQEKYKQEKNVINHPSKEDICATSCEEAV Cow MKFFIFTCLLAVALAKNTMEHVSSSEESI-ISQETYKQEKNMAINPSKENLCSTFCKEVV Goat MKFFIFTCLLAVALAKHKMEHVSSSEEPINIFQEIYKQEKNMAIHPRKEKLCTTSCEEVV *************:**. ********* * * ** ******: .* **.:*:* *:*.* Pig RNIKEVGYASSSSSEESVDIPAENVKVTVEDKHYLKQLEKISQFYQKFPQYLQALYQAQI Cow RNANEEEYSIGSSSEESAEVATEEVKITVDDKHYQKALNEINQFYQKFPQYLQYLYQGPI Goat RNANEEEYSIRSSSEESAEVAPEEIKITVDDKHYQKALNEINQFYQKFPQYLQYPYQGPI ** :* *: ******.:: *::*:**:**** * *::*.*********** **. * Pig VMNPWDQTKTSAYPFIPTVIQSGEELSTSEEPVSSSQEENTKTVDMESMEEFTKKTELTE Cow VLNPWDQVKRNAVPITPTLNR---------EQLSTSEENSKKTVDMESTEVFTKKTKLTE Goat VLNPWDQVKRNAGPFTPTVNR---------EQLSTSEENSKKTIDMESTEVFTKKTKLTE *:*****.* .* *: **: : * :*:*:*:..**:**** * *****:*** Pig EEKNRIKFLNKIKQYYQKFTWPQYIKTVHQKQKAMKPWNHIKTNSYQIIPNLRYF Cow EEKNRLNFLKKISQRYQKFALPQYLKTVYQHQKAMKPWIQPKTKV---IPYVRYL Goat EEKNRLNFLKIISQYYQKFAWPQYLKTVDQHQKAMKRWTQPKTNA---IPYVRYL *****::**: *.* ****: ***:*** *:***** * : **: ** :**:

(Holt et al. 2016)

Phosphoserine clusters ofα-S1-, α-S2 and β-Caseinsbind Ca Phosphate in casein micelle

Precipitation control by caseins

(Farrell et al., 2006; Holt, 2013 )

Species Alcohol stability(%)

Fat globule(µm)

Casein micelle(nm)

α-S1 casein (wt %)

α-S2 casein(wt %)

β-casein(wt %)

κ-casein(wt %)

Cow 75-85 3-4.5 100-140 38 10 40 12

Buffalo 60-69 4-7.5 200-260 40 9 35 12

Goat 60 2.5-3.5 50-200 20 16 41 17

Camel 65 2-3.9 260-300 65 22 9 4

Differences between dairy species

(Park, 2006)

Species Alcohol stability(%)

Fat globule(µm)

Casein micelle(nm)

α-S1 casein (wt %)

α-S2 casein(wt %)

β-casein(wt %)

κ-casein(wt %)

Cow 75-85 3-4.5 100-140 38 10 40 12

Buffalo 60-69 4-7.5 200-260 40 9 35 12

Goat 60 2.5-3.5 50-200 20 16 41 17

Camel 65 2-3.9 260-300 65 22 9 4

Cow 50-200 38 10 40 12

Sheep 50-200 50 + 40 10

Rabbit ? 32 28 26 9

African elephant

350-700 - - 89 11

Human 400-800 3 - 70 27

Horse 150-700 55 + 45 7

Differences between species

(Holt 2012; Madende, 2016)

Casein micelleCow

Size = 240nmSheep fresh milkSize = 50-200nm

Horse fresh milkSize = 150-700nm

Human fresh milk Size = 400-800nm

Elephant fresh milkSize = 350-700nm

Questions

Are all 4 caseins needed to form casein micelle? Are all 4 caseins needed to stabilize casein micelle?

Evolution: First casein –κ-casein- then β-casein

Seems as if only κ- and β-caseins can stabilize- and bind amorphous Ca Phos

Answer

Precipitation control by caseins

(Adapted from Farrell et al., 2006; Holt, 2013 )

CALCIUM PHOSPHATE BINDING

Species α-S1 casein (wt %)

α-S1 casein P-Ser

α-S2 casein(wt %)

α-S1 casein P-Ser

β-casein(wt %)

β-caseinP-Ser

κ-casein(wt %)

κ-caseinP-Ser

TotalP-Ser(calculated)

Cow 38 8 10 3 40 5 12 1 546

Sheep 50 ? + ? 40 ? 10 ? ?

Rabbit 32 ? 28 ? 26 ? 9 ? ?

African elephant

- - - - 89 1 11 1 100

Human 3 4 (1-7) - - 70 4 (1-5) 27 1 202

Horse 55 3 + 3 45 4 (3-7) 7 1 355

Differences between species

• At least 3 P-Ser needed to bind Ca Phosphate• Ratio of casein types - total number of Ca Phosphate• Determines stability of micelle

• Structure aspects of caseins and casein micelles vague• The “vagueness” very obvious when other species

studied• Based on research of bovine casein as well as non-

related proteins

• Different casein type ratios - other species• Precipitation control of other species caseins• Ca Phos binding capacity of micelles

Conclusions

Future

T: +27(0)51 401 9111 | info@ufs.ac.za | www.ufs.ac.za

Danke