MILK PROTEIN POLYMORPHISM

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ADVISORS

Major Advisor: Dr. A.M. ThakerMinor Advisor: Dr. C.G. JoshiSubject Advisor: Dr. D.N. Rank

History

• Studies more than 100 years, • Experimental methods not ever

adequate to verify preliminary hypothesis.

• New analytical methods - benefit to milk protein knowledge

• Electrophoresis, Chromatography etc…

Protein fraction %

Casein �-casein �s1-casein �s2-casein �-casein

2.7 1.1 1.0 0.3 0.3

Whey proteins

�-lactoglobulin (12) �-lactalbumin Immunoglobulins Seric bovine serum albumen Glycomacropeptid

0.70

0.40 0.15 0.10 0.04 0.01

Minor proteins

Lactoferrin #0.01

Enzymes Lactoperoxidase

#0.01

P/ Ca sensitive

-CHO\ Ca resistant

MILK PROTEIN COMPOSITION

Genetic polymorphism

• The term "genetic polymorphism" defines the fact that each milk protein presents two or more forms genetically determined by autosomal and codominant alleles.

• The absence of dominance is very useful, because homozygous individuals present in the electropherogram only one variant for each protein, while heterozygous ones both variants, so that the count of the gene frequencies for a population results very easy.

Genetic polymorphism significance

• to point out milk protein chemical evolution • find some eventual similarity with other

proteins;• to verify relationships between different

species or breeds; • to monitor variations that happen in the time

or in the space for a particular animal • zootechnical sciences

Genetic polymorphism significance

• dairy industry• reproduction efficiency and adaptation capacity, • The relationships between protein genetic types

and milk composition and properties • milks characterized by B variants of �-lactoglobulin, � -casein and b-casein present a nitrogen composition and/or rennet-coagulation properties that are better with respect to those characterized by A types and, therefore, are more favorable for the cheese making.

Nomenclature• The nomenclature of the variants for each

protein: progressive alphabetical order (electrophoretical mobility of the bands).

• The nomenclature is unified for the four species of Bos genus, i.e. B. taurus ("common" bovine), B. indicus (zebu), B. grunniens (yak), B. javanicus (banteng of Bali).

• Some exceptions are made for the progressive alphabetical nomenclature: – different variants are named with the same letter or– on the contrary, the same variant had received two

different "letters"; – there are several variants not well or not still

characterised, for which is not known the aminoacidsubstitution or its position in the molecule

STRUCTURAL ORGANISATION

• More than 95% of the proteins contained in ruminants’ milk are synthesised from 6 structural genes encoding proteins,

• �-lactalbumin and �-lactoglobulin, the two main whey proteins in ruminants, and the four caseins �s1, �s2, � and � which are encoded by four tightly linked and clustered genes

POLYMORPHISMS IN CIS-REGULATORY

ELEMENTS

• Computer aided analysis of the 5’-flanking regions of the milk protein genes allowed the identification of several putative cis regulatory sequences and potential sites of binding of transacting regulatory factors.

• Most of the regulatory sequences are located in the proximal regions of milk protein gene promoters, within 200–250 bp adjacent to the initiation of the transcription site.

• Genetic polymorphisms within the 5’-flanking regions of milk protein genes have been reported

�-lactalbumin• �-lactalbumin is essential for the biosynthesis of lactose

in the mammary gland. • It enhances the substrate affinity of �-1,4

galactosyltransferase which catalyses the formation of lactose from glucose and UDP galactose

• Due to its prominent role in milk synthesis, �-lactalbumin is considered a valuable genetic marker for milk production traits in cattle.

• Bleck and Bremel (1993) found three single-bppolymorphisms in 5’-flanking region of Holstein cattle.

• A single base difference (A/G) at position +15 in the 5’-region of the � lactalbuminm RNA has been shown as a useful marker associated with an increased milk yield in Holstein cows.

• The polymorphism of �-lactalbumin itself has a functional effect or it is linked to some other gene polymorphism that has a functional influence on milk production.

�-lactalbumin

�-lactoglobulin• It is absent from the milk of rodents, rabbits and

camels in which instead another major whey protein the whey acidic protein or WAP is found.

• 12 polymorphic variants of �-lactoglobulin are known in cattle, but the two most frequent, A and B

• These variants differ by two amino acid substitutions in the polypeptide chain arising from two single nucleotide substitutions in the �-lactoglobulin gene: Asp 64 (GAT) to Gly (GGT) and Val 118 (GTC) to Ala (GCC).

• Polymorphism within the 5’-flanking region of the bovine �-lactoglobulin gene (�-lgPTU) was studied in which the two predominant �-lactoglobulin variants are also A and B.

• Moreover, within the promoter region (�-1gP) of the gene, several point mutations were found by SSCP analysis

� - lactoglobulin

� - lactoglobulin

�-casein

• Two major genetic variants of �-casein, A and B, have been identified in cattle.

• Variant A shows Thr (ACC) and Asp (GAT) at positions 136 and 148, respectively,

• B variant shows Ile (ATC) and Ala (GCT) at the same positions.

• These differences are the result of single base mutations in the �-casein gene and the two alleles (A and B) can be distinguished by the presence or absence of a HindIIIrestriction endonuclease recognition site.

• the change in amino acid position 148 abolishes a HinfIsite in the �-casein B allele

• In general, the B variant of �-casein has been recognized as superior for milk quality in European cattle breeds; the B allele of �-casein is associated with shorter renneting time of the milk

• Significant differences have been found in the content of both variants A and B of �-casein in milks of heterozygous AB cows

• Usually there is more of the protein variant encoded by allele A than that encoded by allele B.

• Moreover, seven polymorphic sites have been identified within the 3’-flanking region of the �-casein gene and the differences were shown in the length of mRNAs derived from A and B alleles of the gene.

• The regulation on the post-transcriptional stages of �-casein expression has been suggested as a possible mechanism of differential allelic expression.

• Schild and Geldermann identified a total of 34 variable sites in approx. 1.2 kb 5’-flanking regions of genes encoding bovine calcium-sensitive caseins: 17, 10 and 7 for �s1, �s2 and �-casein, respectively.

�-casein

Discovery of �s1-casein

Discovery of �s2-casein

Discovery of �-casein

Discovery of �-casein

Discovery of �-casein

Discovery of �-casein

POST-TRANSCRIPTIONAL REGULATION

1. Defect in the processing of primary transcripts: alternative splicing

2. Casual usage of cryptic splice sites3. Casual exon-skipping

CONCLUSIONS• to understand the role that each variant can have on milk

nutritional and technological properties.

• to inquire into the causes that determine these effects on the composition and on the coagulation parameters

• to understand why a simple mutation can have such a relevance for the properties of milk

• if properties arise only from the variation in the protein or rather are due to the action of promoters present in the regulatory not coding sequences of DNA.

• important biotechnological applications, like genetic improvement and molecular engineering, with the aim to obtain breeds more and more suitable for the modern requirements.

Thanks…