Anne-Marie Fiat

Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects

SummaryThe first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (σsl-, σs2- β- and κ). The calcium-sensitive casein fractions are always phosphorylated whereas κ-caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca2+. In κ-caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine κ-casein and up to 10 in human κ-casein. The structures of the known κ-casein carbohydate moieties are described. Finally the milk clotting process (interaction κ-casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena.The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A ‘strategic zone’ containing immunostimulating and opioid peptides could be located in cow and human β-caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the κ-caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities.In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components.If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.

Characterization of an antithrombotic peptide from kappa-casein in newborn plasma after milk ingestion.

Bovine and human kappa-caseinoglycopeptides, two antithrombotic peptides derived from the corresponding kappa-caseins, were detected in physiologically active concentrations in the plasma of 5-d-old newborn infants after ingestion of cows-milk-based formula or human milk respectively. It is suggested that these two bioactive peptides are released from milk proteins during digestion.

Sheep κ-casein peptides inhibit platelet aggregation

The C-terminal part (residues 106-171) of sheep kappa-casein, called caseinoglycopeptide (CGP), inhibits thrombin- and collagen-induced platelet aggregation in a dose-dependent manner (mean inhibitory concentration (IC50) 215 microM and 100 microM, respectively). An enzymatic hydrolysate of CGP was fractionated by reverse phase high performance liquid chromatography: three peptides KDQDK (residues 112-116), TAQVTSTEV (residues 163-171) and QVTSTEV (residues 165-171) completely inhibited thrombin-induced platelet aggregation. CGP at a concentration near its IC50 had a very long life when incubated in human or guinea-pig plasma. An ex vivo experiment showed that 17% of CGP was found 60 min after its i.v. bolus injection in guinea-pig. By hydrophobic cluster analysis, human fibrinogen and sheep kappa-casein peptides, inhibitors of platelet aggregation, were compared and we observed similarities for their C-terminal parts and for their short peptides (RGDF and KDQDK).

Effect of κ-casein split peptides on platelet aggregation and on thrombus formation in the guinea-pig

An undecapeptide (residues 106-116 of cow kappa-casein) is known to inhibit human platelet aggregation and fibrinogen binding through inhibition of the interaction between the fibrinogen gamma-chain C-terminus and alphaIIbbeta3. This was due to structural homologies with the fibrinogen gamma-chain C-terminal dodecapeptide. We have therefore compared in this work the in vitro anti-aggregating activity of kappa-casein split peptides and their in vivo potential antithrombotic activity in a model of arterial thrombosis triggered by laser-induced intimal injury in the guinea-pig. Caseinoglycopeptide (residues 106-169), the undecapeptide (residues 106-116) and the pentapeptide KNQDK (residues 112-116) from cow kappa-casein, were anti-aggregating peptides and exerted a significant antithrombotic activity in the guinea-pig. Caseinoglycopeptides from three species (cow, ewe and human) were also antithrombotic and the most potent being the human one. The antithrombotic activity was achieved in vivo for doses less than the one suspected from in vitro data and for which, ex vivo platelet aggregation was not decreased. In conclusion, the relative involvement of the fibrinogen gamma-chain C-terminal dodecapeptide could be much more important in in vivo thrombosis process than in in vitro platelet aggregation. Its specificity and activity in vivo unveiled an interesting potential way for inhibition of arterial thrombosis if alternative molecular presentation (i.e. peptidomimetics) and alternative route (i.e. per os) can be developed.

Isolation and characterization of sheep lactoferrin, an inhibitor of platelet aggregation and comparison with human lactoferrin

Highly purified sheep lactoferrin was isolated from ovine whey in a single chromatographic step (FPLC): it was characterized by electrophoresis, N-terminal sequence determination and compared with lactoferrins from other species. Sheep and human lactoferrins inhibited thrombin-induced platelet aggregation (median inhibitory concentration: IC50 5 and 4 microM, respectively). Pepsin hydrolysates of human and sheep lactoferrins were fractionated by reverse-phase high-performance liquid chromatography and only one peak was an inhibitor of platelet aggregation. The sheep or human lactoferrin binding to platelets was studied.

Biologically active peptides of casein and lactotransferrin implicated in platelet function.

Casein and other milk proteins in maternal colostrum and milk, the earliest food of the newborn, should not only be considered as a nutritional supply but also as a source of biologically active peptides. Some of them isolated from casein and lactotransferrin were active on platelet function. They inhibited both aggregation of ADP-treated platelets and binding of [125I]fibrinogen to ADP-treated platelets. Their behaviour was compared to that of fibrinogen peptides possessing similar effects: once more similarities between the milk and blood-clotting phenomena could be observed.

Binding of the bovine caseinoglycopeptide to the platelet membrane glycoprotein GPIbα

The bovine caseinoglycopeptide (residues 106‐169), the C‐terminal part of κ‐casein, inhibited the von Willebrand factor‐dependent platelet aggregation in a dose‐dependent manner. An affinity matrix made of the caseinoglycopeptide selectively bound the platelet membrane glycoprotein GPIbα which contains the von Willebrand factor binding site. The amino acid residues of GPIbα participating in the caseinoglycopeptide binding were located after residue Glu90.

Immunological cross reactivity between bovine fibrinogen and bovine κ-casein☆

Two coagulation processes occur in nature: the clotting of blood and the clotting of milk. The enzymic digestions of K-casein and fibrinogen, the natural substrates of chymosin (EC 3.4.23.4) and thrombin (EC 3.4.4.13) respectively, present some common features which have been summarized by P. Jolles [l] in a recent review article: high specificity of the enzymes, conservation of the renninand thrombin-sensitive sequences in the course of the evolution, liberation of hydrophilic acid peptides (caseinoglycopeptides or fibrinopeptides) devoid of aromatic amino acids, cyst(e)ine and arginine. Furthermore homology in the amino acid sequences between cow and sheep K-caseins and the /3 and y chains of human fibrinogen has been suggested by J. Jolles et al. [2]. These observations constituted the starting point of a first series of immunological studies concerning a possible cross reactivity between fibrinogen and K-casein.