Hostname: page-component-cd9895bd7-jkksz Total loading time: 0 Render date: 2024-12-23T19:31:24.786Z Has data issue: false hasContentIssue false
  • English
  • Français

Isolation and identification of a high molecular weight protein in sow milk

Published online by Cambridge University Press:  16 January 2015

Y. Qin ,
N. Qi ,
Y. Tang ,
J. He ,
X. Li ,
F. Gu  and
S. Zou
Y. Qin*
Affiliation:
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
N. Qi
Affiliation:
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
Y. Tang
Affiliation:
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
J. He
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
X. Li
Affiliation:
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
F. Gu
Affiliation:
School of Basic Medical Sciences, Anhui Medical University, Hefei 230032, China
S. Zou*
Affiliation:
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
*
†E-mail: [email protected]; [email protected]
†E-mail: [email protected]; [email protected]
Get access

Abstract

A high molecular weight protein (HMWP) was isolated and purified from sow milk, and some of its biochemical characteristics and biological functions were identified. The origin of HMWP was also investigated. The molecular weight of HMWP was determined to be about 115 000 and 114 800 by SDS-PAGE and gel filtration, respectively. The sequence of 10 amino acids in N-terminal of HMWP was Ala-Leu-Val-Gln-Ser-Cys-Leu-Asn-Leu-Val. The sequence was blasted against GenBank. No protein showed significant similarity with this sequence suggesting the HMWP may be novel. The result of liquid chromatography mass spectrometry (LC-MS) also proved HMWP could be a novel protein. By amino acid assay, HMWP was rich in glutamate (including glutamine), cysteine, glycine, aspartic acid (including asparagines) and proline. The content of hydrophobic amino acids (Ala, Val, Leu, Ile, Met, Phe and Pro) was lower at 18.59% of the total amino acids suggesting HMWP has high solubility in water. Western blots of lectins were used to identify the kinds of carbohydrate residues attached to HMWP qualitatively. The result showed that HMWP was a kind of glycoprotein containing N-acetylneuraminic acid (NeuNAc), mannose (Man) and/or N-acetylglucosamine (GlcNAc). By isoelectric focusing, HMWP pI was found to be 5.1. Compared with milk fat globule membrane protein (MFGMP) isolated from the sow milk in SDS-PAGE, MFGMP did not contain HMWP. HMWP was assumed to be a secretory milk protein. HMWP was not found in bovine, goat, rabbit or human milk in SDS-PAGE gel suggesting HMWP may be unique to sow milk. By Western blot, HMWP could be detected in sow milk, not in sow serum, which suggests it is synthesized and secreted by the mammary gland. HMWP concentrations in sows milk were the lowest in the first day of lactation, rose significantly during lactation 1 to 7 days. The HMWP content of sows milk remained relatively constant ((1.95±0.13) g/l) during lactation 7 to 20 days. HMWP significantly inhibited Escherichia coli in a dose related manner in vitro. Overall, HMWP could be a novel sow milk protein with implications for the mammary gland and the piglet.

Keywords

high molecular weight protein (HMWP)sow milkisolationidentificationconcentration
Type
Research Article
Information
animal , Volume 9 , Supplement 5 , May 2015 , pp. 847 - 854
Copyright
© The Animal Consortium 2015 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Andrews, BA, Schmidt, AS and Asenjo, JA 2005. Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. Biotechnology and Bioengineering 90, 380–390.Google Scholar
Bergmann-Leitner, ES, Mease, RM, Duncan, EH, Khan, F, Waitumbi, J and Angov, E 2008. Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies. Malaria Journal 7, 129.Google Scholar
Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.CrossRefGoogle ScholarPubMed
Chan, FL, Poon, HK, Huang, Y and Choi, HL 1999. Glycoconjugates of the rat ciliary body epithelium: a lectin histochemical and protein blotting study. The Histochemical Journal 31, 95–107.CrossRefGoogle ScholarPubMed
Chen, JC, Frankshun, AL, Wiley, AA, Miller, DJ, Welch, KA, Ho, TY, Bartol, FF and Bagnell, CA 2011. Milk-borne lactocrine-acting factors affect gene expression patterns in the developing neonatal porcine uterus. Reproduction 141, 675–683.CrossRefGoogle ScholarPubMed
Crowther, JR 2008. The ELISA guidebook. Humana Press, Totowa, New Jersey.Google Scholar
Demeter, RM, Markiewicz, K, van Arendonk, JA and Bovenhuis, H 2010. Relationships between milk protein composition, milk protein variants, and cow fertility traits in Dutch Holstein-Friesian cattle. Journal of Dairy Science 93, 5495–5502.Google Scholar
Egito, AS, Miclo, L, Lopez, C, Adam, A, Girardet, JM and Gaillard, JL 2002. Separation and characterization of maresʼ milk alpha(s1)-, beta-, kappa-caseins, gamma-casein-like, and proteose peptone component 5-like peptides. Journal of Dairy Science 85, 697–706.Google Scholar
Erat, M, Demir, H and Sakiroglu, H 2005. Purification of glutathione reductase from chicken liver and investigation of kinetic properties. Applied Biochemistry and Biotechnology 125, 127–138.CrossRefGoogle ScholarPubMed
Gusakov, AV, Antonov, AI and Ustinov, BB 2008. N-Glycosylation in Chrysosporium lucknowense enzymes. Carbohydrate Research 343, 48–55.Google Scholar
Hansen, AV, Strathe, AB, Kebreab, E, France, J and Theil, PK 2012. Predicting milk yield and composition in lactating sows: a Bayesian approach. Journal of Animal Science 90, 2285–2298.Google Scholar
He, J, Liu, Y, Zhu, TS, Xie, X, Costello, MA, Talsma, CE, Flack, CG, Crowley, JG, Dimeco, F, Vescovi, AL, Fan, X and Lubman, DM 2011. Glycoproteomic analysis of glioblastoma stem cell differentiation. Journal of Proteome Research 10, 330–338.Google Scholar
Hernandez, A and Harte, FM 2009. Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk. Journal of Dairy Science 92, 5357–5362.CrossRefGoogle ScholarPubMed
Hurley, WL, Grieve, RC, Magura, CE, Hegarty, HM and Zou, S 1993. Electrophoretic comparisons of lactoferrin from bovine mammary secretions, milk neutrophils, and human milk. Journal of Dairy Science 76, 377–387.Google Scholar
Kontopidis, G, Holt, C and Sawyer, L 2004. Invited review: beta-lactoglobulin: binding properties, structure, and function. Journal of Dairy Science 87, 785–796.CrossRefGoogle ScholarPubMed
Krogh, U, Flummer, C, Jensen, SK and Theil, PK 2012. Colostrum and milk production of sows is affected by dietary conjugated linoleic acid. Journal of Animal Science 90 (suppl. 4), 366–368.Google Scholar
Le, TT, van Camp, J, Rombaut, R, van Leeckwyck, F and Dewettinck, K 2009. Effect of washing conditions on the recovery of milk fat globule membrane proteins during the isolation of milk fat globule membrane from milk. Journal of Dairy Science 92, 3592–3603.CrossRefGoogle ScholarPubMed
Liang, M, Chen, VY, Chen, HL and Chen, W 2006. A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy. Talanta 69, 1269–1277.Google Scholar
Pallesen, LT, Andersen, MH, Nielsen, RL, Berglund, L, Petersen, TE, Rasmussen, LK and Rasmussen, JT 2001. Purification of MUC1 from bovine milk-fat globules and characterization of a corresponding full-length cDNA clone. Journal of Dairy Science 84, 2591–2598.CrossRefGoogle ScholarPubMed
Patton, S, Gendler, SJ and Spicer, AP 1995. The epithelial mucin, MUC1, of milk, mammary gland and other tissues. Biochimica et Biophysica Acta 1241, 407–423.Google Scholar
Qin, Y and Zou, S 2003. Dynamic analyses of protein components in Erhualian sow milk. Journal of Biology 20, 9–11.Google Scholar
Qin, Y, Xu, Y, Zou, S and Hurley, WL 2003. The polymorphism of high molecular weight protein in sow milk and its relationship with lactation performance. Journal of Nanjing Agricultural University 26, 64–67.Google Scholar
Qin, Y, Dong, Q, Zou, S and Xu, Y 2004a. Correlation between polymorphism of high molecular weight protein in sow milk and milk growth factors, litter weight gain. Acta Laser Biology Sinica 13, 124–128.Google Scholar
Qin, Y, Xu, Y, Zou, S and Hurley, WL 2004b. The correlation between gene expressive quantities of high molecular weight protein in milk and sow lactation performance. Acta Veterinaria et Zootechnica Sinica 35, 256–259.Google Scholar
Sambrook, J and Russell, DW 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
Wilcox, CP, Janolino, VG and Swaisgood, HE 2002. Isolation and partial characterization of CD36 from skim milk. Journal of Dairy Science 85, 1903–1908.Google Scholar
Wilson, NL, Robinson, LJ, Donnet, A, Bovetto, L, Packer, NH and Karlsson, NG 2008. Glycoproteomics of milk: differences in sugar epitopes on human and bovine milk fat globule membranes. Journal of Proteome Research 7, 3687–3696.CrossRefGoogle ScholarPubMed
Zhang, H, Zou, S, Qin, Y and Chen, W 2004. Homogenous identification of a group of polymorphic high molecular weight protein in sow milk. Scientia Agricultura Sinica 37, 1369–1373.Google Scholar
Zhang, H, Zong, Y, Zou, S, Chen, W and Qin, Y 2006. The evidence of the origin of a polymorphic high molecular weight protein in sow milk. Scientia Agricultura Sinica 39, 798–801.Google Scholar
Zou, S, McLaren, DG and Hurley, WL 1992. Pig colostrum and milk composition: comparisons between Chinese Meishan and US breeds. Livestock Production Science 30, 115–127.Google Scholar
Supplementary material: Image

Qin Supplementary Material

Figure S1

Download Qin Supplementary Material(Image)
Image 1.4 MB
Supplementary material: Image

Qin Supplementary Material

Figure S2

Download Qin Supplementary Material(Image)
Image 4.8 MB
Supplementary material: Image

Qin Supplementary Material

Figure S3

Download Qin Supplementary Material(Image)
Image 939 KB