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Primary structure of ovine αsl-caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D*

Published online by Cambridge University Press:  01 June 2009

Pasquale Ferranti ,
Antonio Malorni ,
Gianpaolo Nitti ,
Pasquale Laezza ,
Rosa Pizzano ,
Lina Chianese  and
Francesco Addeo
Pasquale Ferranti
Affiliation:
Dipartimento di Scienza degli Alimenti,, Facoltà di AgrariaUniversità degli Studi di Napoli Federico II, Parco Gussone, 80055 Portici, Italia
Antonio Malorni
Affiliation:
Servizio di Spettrometria di Massa, CNR, Via Pansini 5, 80131 Napoli, Italia
Gianpaolo Nitti
Affiliation:
CEINGE, Via Pansini 5, 80131 Napoli, Italia
Pasquale Laezza
Affiliation:
Dipartimento di Scienza degli Alimenti,, Facoltà di AgrariaUniversità degli Studi di Napoli Federico II, Parco Gussone, 80055 Portici, Italia
Rosa Pizzano
Affiliation:
Dipartimento di Scienza degli Alimenti,, Facoltà di AgrariaUniversità degli Studi di Napoli Federico II, Parco Gussone, 80055 Portici, Italia
Lina Chianese
Affiliation:
Dipartimento di Scienza degli Alimenti,, Facoltà di AgrariaUniversità degli Studi di Napoli Federico II, Parco Gussone, 80055 Portici, Italia
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Summary

The primary structures of ovine α>s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141–148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for ∼ 80% of the entire translated αsl-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89 % degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in αsl-casein A, whereas Ser115 and Ser41 were phosphorylated by ∼ 50 and ∼ 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13 →> Pro13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP12→>Ser12. A further substitution, SerP68 →> Asn68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser64 and Ser66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.

Type
Original Articles
Information
Journal of Dairy Research , Volume 62 , Issue 2 , May 1995 , pp. 281 - 296
Copyright
Copyright © Proprietors of Journal of Dairy Research 1995

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References

REFERENCES

Addeo, F., Mauriello, R., Moio, L., Laezza, P., Chianese, L. & Di Luccia, A. 1992 Ovine casein variant identification using electrophoretic, immunochemical and chromatographic techniques. Milchwissenshaft 47 283–287Google Scholar
Boisnard, M., Hue, D., Bouniol, C., Mercier, J. -C. & Gaye, P. 1991 Multiple mRNA species code for two non-allelic forms of ovine αs2-casein. European Journal of Biochemistry 201 633–641CrossRefGoogle Scholar
Boisnard, M. & Petrissant, G. 1985 Complete sequence of ovine αs2-casein messenger RNA. Biochimie 67 1043–1051CrossRefGoogle Scholar
Brignon, G., Mahé, M. -F., Grosolaude, F. & Ribadeau-Dumas, B. 1989 Sequence of caprine αsl-casein and characterization of those of its genetic variants which are synthesized at a high level, αsl-Cn A, B and C. Protein Sequences and Data Analysis 2 181–188Google Scholar
Brignon, G., Mahé, M. -F., Ribadeau-Dumas, B., Mercier, J. -C. & Grosclaude, F. 1990 Two of the three genetic variants of goat αsl-casein which are synthesized at a reduced level have an internal deletion possibly due to altered RNA splicing. European Journal of Biochemistry 193 237–241CrossRefGoogle Scholar
Chianese, L., Mauriello, R., Moio, L., Intorcia, N. & Addeo, F. 1992 Determination of ovine casein heterogeneity using gel electrophoresis and immunochemical techniques. Journal of Dairy Research 59 39–47CrossRefGoogle ScholarPubMed
Chianese, L., Mauriello, R., Moio, L., Intorcia, N., Campus, R. & Addeo, F. 1990 [Casein characterization in the Sarda ovine breed.] Proceedings of the XLIVth SJ.S.Vet. (Italian Veterinary Science Society) National Congress, Stresa 44 1701–1704Google Scholar
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. 1989 Electrospray ionization for mass spectrometry of large biomolecules. Science 246 64–71CrossRefGoogle ScholarPubMed
Ferranti, P., Malorni, A., Marino, G., Pucoi, P., Goodwin, G. H, Manfioletti, G. & Gianootti, V. 1992 Mass spectrometric analysis of the HMGY protein from Lewis lung carcinoma: identification of phosphorylation sites. Journal of Biological Chemistry 267 22486–22489CrossRefGoogle ScholarPubMed
Jaubert, A. & Martin, P. 1992 Reverse-phase HPLC analysis of goat caseins. Identification of αsl and αs2 genetic variants. Lait 72 235–247CrossRefGoogle Scholar
Jollès, J., Fiat, A. -M., Schoentgen, F., Alais, C. & Jollès, P. 1974 The amino acid sequence of sheep κA casein. II. Sequence studies concerning the αA-caseinoglycopeptide and establishment of the complete primary structure of the protein. Biochimica et Biophysica Acta 365 335–343CrossRefGoogle Scholar
King, J. W. B. 1967 The caseins of sheep's milk. In Polymorphismes Biochimiques des Animaux, pp. 427–431. Paris: INRA (European Conference on Animal Blood Groups and Biochemical Polymorphisms no. 10 1966)Google Scholar
Leroux, C., Mazure, N. & Martin, P. 1992 Mutations away from splice site recognition sequences might cismodulate alternative splicing of goat αsl-casein transcripts. Structural organization of the relevant gene. Journal of Biological Chemistry 267 6147–6157CrossRefGoogle Scholar
Mcknight, R. A., Jiminez-Flores, R., Kang, Y., Creamer, L. K. & Richardson, T. 1989 Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine αsl-casein A from mammary tissue of a heterozygous B variant cow. Journal of Dairy Science 72 2464–2473CrossRefGoogle Scholar
Manson, W., Carolan, T. & Annan, W. D. 1977 Bovine αs0-easein, a phosphorylated homologue of αsl-casein. European Journal of Biochemistry 78 411–417CrossRefGoogle Scholar
Mercier, J. -C. 1981 Phosphorylation of caseins. Present evidence for an amino acid triplet code posttranslationally recognized by specific kinases. Biochimie 63 1–17CrossRefGoogle ScholarPubMed
Mercier, J. -C, Brignon, G. & Ribadeau-Dumas, B. 1973 [Primary structure of bovine κ-casein B. Complete sequence.] European Journal of Biochemistry 35 222–235CrossRefGoogle ScholarPubMed
Mercier, J. -C., Gaye, P., Soulier, S., Hue-Delhaie, D. & Vilotte, J. -L. 1985 Construction and identification of recombinant plasmids carrying cDNAs coding for ovine αsl-, αs2, β-, κ-casein and β- lactoglobulin. Nucleotide sequence of αsl-casein cDNA. Biochimie 67 959–971CrossRefGoogle Scholar
Mercier, J. -C., Grosclaude, F. & Ribadeau-Dumas, B. 1971 [Primary structure of bovine αsl-casein. Complete sequence.] European Journal of Biochemistry 23 41–51CrossRefGoogle Scholar
Meyer, H. E., Hoffmann-Posorske, E., Korte, H. & Heilmeyer, L. M. G. 1986 Sequence analysis of phosphoserine-containing peptides. Modification for picomolar sensitivity. FEBS Letters 204 61–66CrossRefGoogle ScholarPubMed
Morris, H. R., Panico, M. & Taylor, G. W. 1983 FAB-mapping of recombinant-DNA protein products. Biochemical and Biophysical Research Communications 117 299–305CrossRefGoogle ScholarPubMed
Naylor, S., Findeis, A. F., Gibson, B. W. & Williams, D. H. 1986 An approach toward the complete fast atom bombardment analysis of enzymatic digests of peptides and proteins. Journal of the American Chemical Society 108 6359–6363CrossRefGoogle Scholar
Petrilli, P., Pucci, P., Morris, H. R. & Addeo, F. 1986 Assignment of phosphorylation sites in buffalo β-casein by fast atom bombardment mass spectrometry. Biochemical and Biophysical Research Communications 140 28–37CrossRefGoogle ScholarPubMed
Pucci, P., Carestia, C., Fioretti, G., Mastrobuoni, A. M. & Pagano, L. 1985 Protein fingerprint by fast atom bombardment mass spectrometry: characterization of normal and variant human hemoglobins. Biochemical and Biophysical Research Communications 130 84–90CrossRefGoogle Scholar
Richardson, B. G. & Mercier, J. -C. 1979 The primary structure of the ovine β-caseins. European Journal of Biochemistry 99 285–297Google ScholarPubMed
Swaisgood, H. E. 1992 Chemistry of the caseins. In Advanced Dairy Chemistry—1. Proteins, pp. 63–110 (Ed. Fox., P. F.) London: Elsevier Applied ScienceGoogle Scholar