ABC  Vol.2 No.3 , August 2012
A proteomic approach to investigate the qualitative and quantitative polymorphism of β-lactoglobulin in ovine milk: Inference on gene copy-number variations
The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk; 2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene; 3) the β-LG gene is duplicated in the dog and bovine genome; 4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.

Cite this paper
Picariello, G. , Luccia, A. , Ferranti, P. , Alloggio, I. , Addeo, F. and Pieragostini, E. (2012) A proteomic approach to investigate the qualitative and quantitative polymorphism of β-lactoglobulin in ovine milk: Inference on gene copy-number variations. Advances in Biological Chemistry, 2, 207-217. doi: 10.4236/abc.2012.23025.
[1]   Bawden, W.S., Passey, R.J. and Mackinlay, G. (1994) The genes encoding the major milk-specific proteins and their use in transgenic studies and protein engineering. Biotechnology & Genetic Engineering Review, 12, 89- 137.

[2]   Ferranti, P., Mamone, G., Picariello, G. and Addeo, F. (2011) The “dark side” of β-lactoglobulin: Unedited structural features suggest unexpected functions. Journal of Chromatography A, 1218, 3423-3431. Hdoi:10.1016/j.chroma.2011.03.059

[3]   Kontopidis, G., Holt, C. and Sawyer, L. (2004) Invited review: β-lactoglobulin: Binding properties, structure, and function. Journal of Dairy Science, 87, 785-796. Hdoi:10.3168/jds.S0022-0302(04)73222-1

[4]   Liberatori, J. (1977). β-lactoglobulin: Chemical and stru- ctural studies. Folia Veterinaria Latina, 7, 205-222.

[5]   Bell, K. and Mckenzie, H.A. (1967) The whey proteins of ovine milk: β-lactoglobulins A and B. Biochimica et Biophysica Acta, 147, 123-134. Hdoi:10.1016/0005-2795(67)90095-5

[6]   King, J.B.W. (1969) The distribution of sheep beta lacto- globulin. Animal Production, 11, 53-57. Hdoi:10.1017/S0003356100026611

[7]   Erhardt, G. (1989) Evidence for a third allele at the β- lactoglobulin (β-Lg) locus of sheep milk and its occu- rrence in different breeds. Animal Genetics, 20, 197-204. Hdoi:10.1111/j.1365-2052.1989.tb00857.x

[8]   Gaye, P., Hue-Delahaie, D., Mercier, J.C., Soulier, S., Vilotte, J.L. and Furet, J.P. (1986) Ovine beta-lactoglo-bulin messenger RNA: Nucleotide sequence and mRNA levels during functional differentiation of the mammary gland. Biochimie, 68, 1097-1107. Hdoi:10.1016/S0300-9084(86)80184-5

[9]   Erhardt, G., Godovac-Zimmermann, J. and Conti, A. (1989) Isolation and complete primary sequence of a new ovine wild-type ?-lactoglobulin C. Biological Chemistry Hoppe-Seyler, 370, 757-762. Hdoi:10.1515/bchm3.1989.370.2.757

[10]   Amigo, L., Recio, I. and Ramos, M. (2000) Genetic po- lymorphism of ovine milk proteins: Its influence on technological properties of milk-a review. Dairy Journal, 10, 135-149. Hdoi:10.1016/S0958-6946(00)00034-0

[11]   Ehrmann, S., Bartenschlager, H. and Geldermann, H. (1997) Polymorphism in the 5’-flanking region of the bovine β-lactoglobulin-encoding gene and its association with β-lactoglobulin in the milk. Journal of Animal Breeding and Genetics, 114, 49-53. Hdoi:10.1111/j.1439-0388.1997.tb00491.x

[12]   Kaminski, S. and Zabolewicz, T. (2000) Associations between bovine beta-lactoglobulin polymorphism within coding and regulatory sequences and milk performance traits. Journal of Applied Genetics, 41, 91-99.

[13]   Dove, P. (2002) Genetic polymorphisms in milk protein genes and their impact on milk composition. Advances in Experimental Medicine and Biology, 480, 225-230. Hdoi:10.1007/0-306-46832-8_28

[14]   Lemay, D.G., Lynn, D.J., Martin, W.F. et al. (2009) The bovine lactation genome: Insights into the evolution of mammalian milk. Genome Biology, 10, R43. Hdoi:10.1186/gb-2009-10-4-r43

[15]   Rullo, R., Di Luccia, A., Chianese, L. and Pieragostini, E. (2010) Hot topic: Gene duplication at the α-lactalbumin locus: Finding the evidence in water buffalo (Bubalus bubalus L.). Journal of Dairy Science, 93, 2161-2167. Hdoi:10.3168/jds.2009-2627

[16]   Rullo, R., Di Luccia, A., Alloggio, I. and Pieragostini, E. (2010a) Duplicazione al locus dell’alfa lattoalbumina nei ruminanti: Evidenze negli ovini. Proceedings of National Congresso of the Italian Society of Sheep and Goat Pathology and Production (SIPAOC), Pesaro, 22-25 September 2010.

[17]   Rullo, R., Di Luccia, A. and Pieragostini, E. (2010) Evidence of gene duplication at the alpha-lactalbumin locus in cattle (Bos bovis, L.). Proceedings of the 26th World Buiatrics Congress, Santiago, 14-18 November 2010.

[18]   Liu G.E., Hou, Y., Zhu, B., et al. (2010) Analysis of copy number variations among diverse cattle breeds. Genome Research, 20, 693-703. Hdoi:10.1101/gr.105403.110

[19]   Passey, R.J. and Mackinlay, A.G. (1995) Characterisation of a second, apparently inactive, copy of the bovine beta- lactoglobulin gene. European Journal of Biochemistry, 233, 736-743. Hdoi:10.1111/j.1432-1033.1995.736_3.x

[20]   Ohno, S. (1984) Birth of a unique enzyme from an alternative reading frame of the pre-existed, internally repetitious coding sequence, Proceedings of the National Academy of Sciences USA, 81, 2421-2425. Hdoi:10.1073/pnas.81.8.2421

[21]   Folch, J.M., Colla, A., Hayesb, H.C. and Sànchez, A. (1996) Characterization of a caprine β-Lactoglobulin pse- udogene, identification and chromosomal localization by in situ hybridization in goat, sheep and cow. Gene, 177, 87-91. Hdoi:10.1016/0378-1119(96)00276-4

[22]   Hatje, K. and Kollmar, M. (2011). Predicting tandemly arrayed gene duplicates with WebScipio. In: Friedberg, F. Ed., Gene Duplication, InTech, Rijeka, 59-76. Hdoi:10.5772/24240

[23]   Aloni, R., Olender, T. and Lancet, D. (2006) Ancient genomic architecture for mammalian olfactory receptor clusters. Genome Biology, 7, R88. Hdoi:10.1186/gb-2006-7-10-r88

[24]   Garcia-Fernandez, J. (2005) The genesis and evolution of homeobox gene clusters. Nature reviews. Genetics, 6, 881-892. Hdoi:10.1038/nrg1723

[25]   Zhang, J. and Nei, M. (1996) Evolution of Antennapediaclass homeobox genes. Genetics, 142, 295-303.

[26]   Proudfoot, N.J. (1986) Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation. Nature, 322, 562-565. Hdoi:10.1038/322562a0

[27]   Vestri, R., Pieragostini, E., Yang, F., Di Gregorio, P., Rando, A. and Masina, P. (1991) Expression of triplicated and quadruplicated alpha globin genes in sheep. British Journal of Haematology, 77, 110-116. Hdoi:10.1111/j.1365-2141.1991.tb07956.x

[28]   Aschaffenburg, R. and Drewry, J. (1959) New pro- cedure for the routine determination of the various non- casein proteins of milk. Proceedings of the 15th International Dairy Congress, 29 June-3 July 1959, London, 1631-1637.

[29]   Raymond, M. and Rousset, F. (1995) GENEPOP (v 1.2): A population genetics software for exact tests and ecumenicism. Journal of Heredity, 86, 248-249.

[30]   Broad, T.E., Dolling, C.H.S., Lauvergne, J.J. and Millar, P. (1999) Revised COGNOSAG guidelines for gene nomenclature in ruminants. Genetic Selection Evolution, 31, 263-268. Hdoi:10.1186/1297-9686-31-3-263

[31]   Vestri, R., Pieragostini, E. and Ristaldi, M.S. (1994) Expression gradient in sheep alpha alpha and alpha alpha alpha globin gene haplotypes: mRNA levels. Blood, 83, 2317-2322.

[32]   Pieragostini, E., Petazzi, F. and Di Luccia, A. (2003) The relationship between the presence of extra alpha-globin genes and blood cell traits in Altamurana sheep. Genetic Selection Evolution, 35, S121-S133. Hdoi:10.1186/1297-9686-35-S1-S121

[33]   Lum L.S., Dovc, P. and Medrano, J.F. (1997) Polymorphisms of bovine beta-lactoglobulin promoter and differences in the binding affinity of activator protein-2 transcription factor. Journal of Dairy Science, 80, 1389-1397. Hdoi:10.3168/jds.S0022-0302(97)76068-5

[34]   Braunschweig M.H. and Leeb, T. (2006) Aberrant low expression level of bovine beta-lactoglobulin is associ- ated with a C to A transversion in the BLG promoter region. Journal of Dairy Science, 89, 4414-4419. Hdoi:10.3168/jds.S0022-0302(06)72488-2

[35]   Karim, S.A., Barrie, J.A., McCulloch, M.C., et al. (2007) PLP overexpression perturbs myelin protein composition and myelination in a mouse model of Pelizaeus-Merz-bacher disease. Glia, 55, 341-351. Hdoi:10.1002/glia.20465

[36]   Regis, S., Grossi, S., Corsolini, F., Biancheri R. and Filocamo, M. (2009) PLP1 gene duplication causes overex- pression and alteration of the PLP/DM20 splicing balance in fibroblasts from Pelizaeus-Merzbacher disease patients. Biochimica et Biophysica Acta, 1792, 548-554. Hdoi:10.1016/j.bbadis.2009.04.002

[37]   Pieragostini, E., Alloggio, I. and Petazzi, F. (2010) In- sights into hemoglobin polymorphism and related func- tional effects on hematological pattern in mediterranean cattle, goat and sheep. Diversity, 2, 679-700. Hdoi:10.3390/d2040679

[38]   Cubric-CuriK V., Feligini, M., Lukac-Havranek, J., Curik, I. and Enne, G. (2002) Genetic polymorphism of β-lacto-globulin in native sheep from the island of Pag. Food Technology and Biotechnology, 40, 75-78.

[39]   Giaccone, P., Di Stasio, L., Fiandra, P. and Finocchiaro, R. (1997) Effetto della β-lattoglobulina sulla produzione quanti-qualitativa del latte. Proceedings of the XII the National Congress of Scientific Association of Animal Production (ASPA), 23-26 June 1997, Pisa, 271-272.

[40]   Chiofalo, L., Micari, P. and Girmenia, A.M. (1986) Polimorfismo genetico del locus β-lattoglobulina nella razza ovina Comisana allevate in Sicilia. Zootecnica e Nutri- zione Animale, 12, 73-80.

[41]   Iorio M., Vincenti, D., Annunziata, M., Rullo, R., Bonamassa, M., Di Luccia, A. and Pieragostini, E. (2004) Biochemical and molecular investigations on qualitative and quantitative Hb polymorphism in the river buffalo (Bubalus bubalis L.) population reared in Southern Italy. Genetics and Molecular Biology, 27, 167-173. Hdoi:10.1590/S1415-47572004000200007

[42]   Scaloni, A., Pieragostini, E., Malorni, A., Ferrara, L. and Di Luccia, A. (1998) Bovine hemoglobin alpha-globin chain polymorphism: Primary structure determination of two new genetic variants by mass spectrometry and amino acid sequencing. Biochimie, 80, 333-338. Hdoi:10.1016/S0300-9084(98)80076-X

[43]   Pieragostini, E., Di Luccia, A., Rullo, R. and Bottiglieri, C. (2002) Hemoglobin phenotypes in Murgese horse. Italian Journal of Animal Science, 1, 159-163.

[44]   Sugino R. and Innan H. (2006) Selection for more of the same product as a force to enhance concerted evolution of duplicated genes. Trends in Genetics, 22, 642-644. Hdoi:10.1016/j.tig.2006.09.014

[45]   Zhang, J. (2003) Evolution by gene duplication: An update. Trends in Ecology and Evolution, 18, 292-298. Hdoi:10.1016/S0169-5347(03)00033-8

[46]   Zimmer, E.A., Martin, S.L., Beverley, S.M., Kan, Y.W. and Wilson, A.C. (1980) Rapid duplication and loss of genes coding for the α-chains of hemoglobin. Proceedings of the National Academy of Sciences USA, 77, 2158- 2162. Hdoi:10.1073/pnas.77.4.2158

[47]   Lam, K.-W. L. and Jeffreys, A. (2006) Processes of copy- number change in human DNA: The dynamics of α-globin gene deletion. Proceedings of the National Academy of Sciences USA, 103, 8921-8927.

[48]   Hoffmann, F.G., Opazo J.C. and Storz, J.F. (2008) Rapid rates of lineage-speci?c gene duplication and deletion in the α-globin gene family. Molecular Biology and Evolution, 25, 591-602. Hdoi:10.1093/molbev/msn004

[49]   Hurst, L.D. and Smith, N.G.C. (1998) The evolution of concerted evolution. Proceedings of the Royal Society B: Biological Sciences, 265, 121-127. Hdoi:10.1098/rspb.1998.0272

[50]   Nei, M., Rogozin, I.B. and Piontkivska, H. (2000) Purifying selection and birth-and-death evolution in the ubiquitin gene family. Proceedings of the National Academy of Sciences USA, 97, 10866-10871. Hdoi:10.1073/pnas.97.20.10866

[51]   Piontkivska, H., Rooney, A.P. and Nei, M. (2002) Purifying selection and birth-and-death evolution in the histone H4 gene family. Molecular Biology and Evolution, 19, 689-697. Hdoi:10.1093/oxfordjournals.molbev.a004127

[52]   Higgs D.R., Vickers M.A., Wilkie A.O., Pretorius, I.M., Jarman, A.P. and Weatherall, D.J. (1989) A review of the molecular genetics of the human alpha-globin gene cluster. Blood, 73, 1081-1104.

[53]   Hardison, R.C. (2008) Globin genes on the move. Journal of Biology, 7, 35. Hdoi:10.1186/jbiol92

[54]   Schrider, D.R. and Hahn, M.W. (2010) Gene copy-number polymorphism in nature. Proceedings of the Royal Society B: Biological Sciences, 277, 3213-3221. Hdoi:10.1098/rspb.2010.1180