1. State Key Laboratory of Medicinal Chemical Biology (Nankai University), Tianjin 300071, China; 2. Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, 201210, China; 3. Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin 300071, China; 4. Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China
Abstract：Lactoferrin is an iron-binding glycoprotein with a molecular weight of about 80 kDa that belongs to the transferrin family. Due to its unique physical and chemical properties, lactoferrin has a variety of biological functions including antibacterial, antiviral, anticancer, immunomodulatory activities and regulation of iron absorption. High-yield production of recombinant lactoferrin with biological activity and its application in clinical treatment have been a hot topic for long time. With the development of genetic engineering techniques, various expression systems have been developed to produce recombinant lactoferrin. In this review, we summarize physicochemical characteristics, biological activities, clinical studies and current recombinant expression systems of lactoferrin, in order to provide references for its clinical application.
Johanson B. Isolation of an iron-containing red protein from human milk. Acta Chem Scandinav , 1960, 14(2): 510-512.
[27]
Mata L, Sánchez L, Headon DR, Calvo M. Thermal denaturation of human lactoferrin and its effecton the ability to bind iron. J Agric Food Chem , 1998, 46(10): 3964-3970.
[28]
Bellamy W, Takase M, Wakabayashi H, Kawase K, Tomita M. Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. J Appl Bacteriol , 1992, 73(6): 472-479.
van Berkel PH, van Veen HA, Geerts ME, De Boer HA, Nuijens JH. Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants. Biochem J , 1996, 319(Pt1): 117-122.
[31]
Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN. Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution. J Mol Biol , 1997, 274(2): 222-236.
[32]
Adlerova L, Bartoskova A, Faldyna M. Lactoferrin: a review. Vet Med , 2008, 53(9): 457-468.
[33]
Fleet JC. A new role for lactoferrin: DNA binding and transcription activation. Nutr Rev , 1995, 53(8): 226-227.
[34]
He JL, Furmanski P. Sequence specificity and transcriptional activation in the binding of lactoferrin to DNA. Nature , 1995, 373(6516): 721-724.
[35]
Liao YL, Jiang RL, Lönnerdal B. Biochemical and molecular impacts of lactoferrin on small intestinal growth and development during early life. Biochem Cell Biol , 2012, 90(3): 476-484.
[36]
Lönnerdal B. Infant formula and infant nutrition: bioactive proteins of human milk and implications for composition of infant formulas. Am J Clin Nutr , 2014, 99(3): 712S-717S.
[37]
Suzuki YA, Lopez V, Lönnerdal B. Mammalian lactoferrin receptors: structure and function. Cell Mol Life Sci , 2005, 62(22): 2560-2575.
[38]
Cox TM, Mazurier J, Spik G, Montreuil J, Peters TJ. Iron binding proteins and influx of iron across the duodenal brush border: Evidence for specific lactotransferrin receptors in the human intestine. Biochim Biophys Acta , 1979, 588(1): 120-128.
[39]
Masson PL, Heremans JF, Prignot JJ, Wauters G. Immunohistochemical localization and bacteriostatic properties of an iron-binding protein from bronchial mucus. Thorax , 1966, 21(6): 538-544.
[40]
Majerle A, Kidric J, Jerala R. Enhancement of antibacterial and lipopolysaccharide binding activities of a human lactoferrin peptide fragment by the addition of acyl chain. J Antimicrob Chemother , 2003, 51(5): 1159-1165.
[41]
Teraguchi S, Wakabayashi H, Kuwata H, Yamauchi K, Tamura Y. Protection against infections by oral lactoferrin: evaluation in animal models. Biometals , 2004, 17(3): 231-234.
[42]
Sherman MP, Bennett SH, Hwang FF, Yu C. Neonatal small bowel epithelia: enhancing anti-bacterial defense with lactoferrin and lactobacillus GG. Biometals , 2004, 17(3): 285-289.
[43]
Drago-Serrano ME, Rivera-Aguilar V, Reséndiz-Albor AA, Campos-Rodríguez R. Lactoferrin increases both resistance to Salmonella typhimurium infection and the production of antibodies in mice. Immunol Lett , 2010, 134(1): 35-46.
[44]
Orsi N. The antimicrobial activity of lactoferrin: current status and perspectives. Biometals , 2004, 17(3): 189-196.
[45]
Arias M, McDonald LJ, Haney EF, Nazmi K, Bolscher JGM, Vogel HJ. Bovine and human lactoferricin peptides: chimeras and new cyclic analogs. Biometals , 2014, 27(5): 935-948.
[46]
Bellamy W, Takase M, Yamauchi K, Wakabayashi H, Kawase K, Tomita M. Identification of the bactericidal domain of lactoferrin. Biochim Biophys Acta , 1992, 1121(1-2): 130-136.
[47]
Seganti L, Di Biase AM, Marchetti M, Pietrantoni A, Tinari A, Superti F. Antiviral activity of lactoferrin towards naked viruses. Biometals , 2004, 17(3): 295-299.
[48]
Arnold D, Di Biase AM, Marchetti M, Pietrantoni A, Valenti P, Seganti L, Superti F. Antiadenovirus activity of milk proteins: lactoferrin prevents viral infection. Antiviral Res , 2002, 53(2): 153-158.
[49]
Ishikawa H, Awano N, Fukui T, Sasaki H, Kyuwa S. The protective effects of lactoferrin against murine norovirus infection through inhibition of both viral attachment and replication. Biochem Biophys Res Commun , 2013, 434(4): 791-796.
[50]
Wong JH, Liu ZK, Law KWK, Liu F, Xia LX, Wan DCC, Ng TB. A study of effects of peptide fragments of bovine and human lactoferrins on activities of three key HIV-1 enzymes. Peptides , 2014, 62: 183-188.
Hasegawa K, Motsuchi W, Tanaka S, Dosako S. Inhibition with lactoferrin of in vitro infection with human herpes virus. Jpn J Med Sci Biol , 1994, 47(2): 73-85.
[53]
Picard-Jean F, Bouchard S, Larivée G, Bisaillon M. The intracellular inhibition of HCV replication represents a novel mechanism of action by the innate immune Lactoferrin protein. Antiviral Res , 2014, 111: 13-22.
[54]
Hayes TG, Falchook GF, Varadhachary GR, Smith DP, Davis LD, Dhingra HM, Hayes BP, Varadhachary A. Phase I trial of oral talactoferrinalfa in refractory solid tumors. Invest New Drugs , 2006, 24(3): 233-240.
[55]
Varadhachary A, Wolf JS, Petrak K, O'Malley BW, Spadaro M, Curcio C, Forni G, Pericle F. Oral lactoferrin inhibits growth of established tumors and potentiates conventional chemotherapy. Int J Cancer , 2004, 111(3): 398-403.
[56]
McKeown ST, Lundy FT, Nelson J, Lockhart D, Irwin CR, Cowan CG, Marley JJ. The cytotoxic effects of human neutrophil peptide-1 (HNP1) and lactoferrin on oral squamous cell carcinoma (OSCC) in vitro . Oral Oncol , 2006, 42(7): 685-690.
[57]
Sakai T, Banno Y, Kato Y, Nozawa Y, Kawaguchi M. Pepsin-digested bovine lactoferrin induces apoptotic cell death with JNK/SAPK activation in oral cancer cells. J Pharmacol Sci , 2005, 98(1): 41-48.
[58]
González-Chávez SA, Arévalo-Gallegos S, Rascón-Cruz Q. Lactoferrin: structure, function and applications. Int J Antimicrob Agents , 2009, 33(4): 301.e1-8.
[59]
Zhou YH, Zeng ZY, Zhang WL, Xiong W, Wu MH, Tan YX, Yi W, Xiao L, Li XL, Huang C, Cao L, Tang K, Li XY, Shen SR, Li GY. Lactotransferrin: a candidate tumor suppressor-deficient expression in human nasopharyngeal carcinoma and inhibition of NPC cell proliferation by modulating the mitogen-activated protein kinase pathway. Int J Cancer , 2008, 123(9): 2065-2072.
[60]
Wang YZ, Shan TZ, Xu ZR, Feng J, Wang ZQ. Effects of the lactoferrin (LF) on the growth performance, intestinal microflora and morphology of weanling pigs. Anim Feed Sci Technol , 2007, 135(3-4): 263-272.
Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K. Twenty-five years of research on bovine lactoferrin applications. Biochimie , 2009, 91(1): 52-57.
[63]
Sfeir RM, Dubarry M, Boyaka PN, Rautureau M, Tomé D. The mode of oral bovine lactoferrin administration influences mucosal and systemic immune responses in mice. J Nutr , 2004, 134(2): 403-409.
[64]
Fischer R, Debbabi H, Dubarry M, Boyaka P, Tomé D. Regulation of physiological and pathological Th1 and Th2 responses by lactoferrin. Biochem Cell Biol , 2006, 84(3): 303-311.
[65]
Mizuhashi F, Koide K, Toya S, Takahashi M, Mizuhashi R, Shimomura H. Levels of the antimicrobial proteins lactoferrin and chromogranin in the saliva of individuals with oral dryness. J Prosthet Dent , 2015, 113(1): 35-38.
[66]
Manzoni P, Meyer MP, Stolfi I, Rinaldi M, Cattani S, Pugni L, Romeo MG, Messner H, Decembrino L, Laforgia N, Vagnarelli F, Memo L, Bordignon L, Maule M, Gallo E, Mostert M, Quercia M, Bollani L, Pedicino R, Renzullo L, Betta P, Ferrari F, Alexander T, Magaldi R, Farina D, Mosca F, Stronati M. Bovine lactoferrin supplementation for prevention of necrotizing enterocolitis in very-low-birth-weight neonates: a randomized clinical trial. Early Hum Dev , 2014, 90(S1): S60-S65.
[67]
Turin CG, Zea-Vera A, Pezo A, Cruz K, Zegarra J, Bellomo S, Cam L, Llanos R, Castañeda A, Tucto L, Ochoa TJ. Lactoferrin for prevention of neonatal sepsis. Biometals , 2014, 27(5): 1007-1016.
[68]
Kozu T, Iinuma G, Ohashi Y, Saito Y, Akasu T, Saito D, Alexander DB, Iigo M, Kakizoe T, Tsuda H. Effect of orally administered bovine lactoferrin on the growth of adenomatous colorectal polyps in a randomized, placebo-controlled clinical trial. Cancer Prev Res , 2009, 2(11): 975-983.
[69]
García-Montoya I, Salazar-Martínez J, Arévalo-Gallegos S, Sinagawa-García S, Rascón-Cruz Q. Expression and characterization of recombinant bovine lactoferrin in E. coli . BioMetals , 2013, 26(1): 113-122.
Chen HL, Lai YW, Chen CS, Chu TW, Lin W, Yen CC, Lin MF, Tu MY, Chen CM. Probiotic Lactobacillus casei expressing human lactoferrin elevates antibacterial activity in the gastrointestinal tract. BioMetals , 2010, 23(3): 543-554.
[72]
Liang QW, Richardson T. Expression and characterization of human lactoferrin in yeast Saccharomyces cerevisiae . J Agri Food Chem , 1993, 41(10): 1800-1807.
[73]
Jiang TM, Chen LJ, Jia SQ, Chen LS, Ma Y. High-level expression and production of human lactoferrin in Pichia pastoris . Dairy Sci Technol , 2008, 88(2): 173-181.
[74]
Paramasivam M, Saravanan K, Uma K, Sharma S, Singh TP, Srinivasan A. Expression, purification, and characterization of equine lactoferrin in Pichia pastoris . Protein Expr Purif , 2002, 26(1): 28-34.
[75]
Wang SH, Yang TS, Lin SM, Tsai MS, Wu SC, Mao SJ. Expression, characterization, and purification of recombinant porcine lactoferrin in Pichia pastoris . Protein Expr Purif , 2002, 25(1): 41-49.
[76]
Li JB, Zhu WZ, Luo MR, Ren HH, Tang L, Liao HH, Wang Y. Molecular cloning, expression and purification of lactoferrin from Tibetan sheep mammary gland using a yeast expression system. Protein Expr Purif , 2015, 109: 35-39.
[77]
Ward PP, Lo JY, Duke M, May GS, Headon DR, Conneely OM. Production of biologically active recombinant human lactoferrin in Aspergillus oryzae . Nat Biotechnol , 1992, 10(7): 784-789.
[78]
Ward PP, May GS, Headon DR, Conneely OM. An inducible expression system for the production of human lactoferrin in Aspergillus nidulans . Gene , 1992, 122(1): 219-223.
[79]
Ward PP, Piddington CS, Cunningham GA, Zhou XD, Wyatt RD, Conneely OM. A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic. Nat Biotechnol , 1995, 13(5): 498-503.
[80]
Mitra A, Zhang Z. Expression of a human lactoferrin cDNA in tobacco cells produces antibacterial protein(s). Plant Physiol , 1994, 106(3): 977-981.
[81]
Salmon V, Legrand D, Slomianny MC, El Yazidi I, Spik G, Gruber V, Bournat P, Olagnier B, Mison D, Theisen M, Mérot B. Production of human lactoferrin in transgenic tobacco plants. Protein Expr Purif , 1998, 13(1): 127-135.
[82]
Chong DKX, Langridge WHR. Expression of full-length bioactive antimicrobial human lactoferrin in potato plants. Transgenic Res , 2000, 9(1): 71-78.
[83]
Min SR, Woo JW, Jeong WJ, Han SK, Lee YB, Liu JR. Production of human lactoferrin in transgenic cell suspension cultures of sweet potato. Biol Plant , 2006, 50(1): 131-134.
[84]
Nandi S, Suzuki YA, Huang JM, Yalda D, Pham P, Wu LY, Bartley G, Huang N, Lönnerdal B. Expression of human lactoferrin in transgenic rice grains for the application in infant formula. Plant Sci , 2002, 163(4): 713-722.
[85]
Stowell KM, Rado TA, Funk WD, Tweedie JW. Expression of cloned human lactoferrin in baby-hamster kidney cells. Biochem J , 1991, 276 (Pt2): 349-355.
[86]
Platenburg GJ, Kootwijk EPA, Kooiman PM, Woloshuk SL, Nuijens JH, Krimpenfort PJA, Pieper FR, de Boer HA, Strijker R. Expression of human lactoferrin in milk of transgenic mice. Transgenic Res , 1994, 3(2): 99-108.
[87]
Nuijens JH, van Berkel PHC, Geerts MEJ, Hartevelt PP, de Boer HA, van Veen HA, Pieper FR. Characterization of recombinant human lactoferrin secreted in milk of transgenic mice. J Biol Chem , 1997, 272(13): 8802-8807.
[88]
Liu ZL, Zhao CJ, Fan BL, Dai YP, Zhao ZH, Wang LL, Zheng M, Feng JD, Chen YZ, Duan YL, Li N. Variable expression of human lactoferrin gene in mice milk driven by its 90 kb upstream flanking sequences. Anim Biotechnol , 2004, 15(1): 21-31.
[89]
van Berkel PHC, Welling MM, Geerts M, van Veen HA, Ravensbergen B, Salaheddine M, Pauwels EKJ, Pieper F, Nuijens JH, Nibbering PH. Large scale production of recombinant human lactoferrin in the milk of transgenic cows. Nat Biotechnol , 2002, 20(5): 484-487.
[90]
Cui D, Li J, Zhang LL, Liu S, Wen X, Li QY, Zhao YF, Hu XX, Zhang R, Li N. Generation of bi-transgenic pigs overexpressing human lactoferrin and lysozyme in milk. Transgenic Res , 2015, 24(2): 365-373.
[91]
Yu HQ, Chen JQ, Sun W, Liu SG, Zhang AM, Xu XJ, Wang XB, He ZZ, Liu GH, Cheng GX. The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct. J Biotechnol , 2012, 161(3): 198-205.
[92]
Nuc P, Nuc K. Recombinant protein production in Escherichia coli . Postepy Biochem , 2005, 52(4): 448-456.
[93]
Dong XB, Tang B, Li J, Xu Q, Fang ST, Hua ZC. Expression and purification of intact and functional soybean ( Glycine max ) seed ferritin complex in Escherichia coli . J Microbiol Biotechnol , 2008, 18(2): 299-307.
[94]
Yurimoto H. Molecular basis of methanol-inducible gene expression and its application in the methylotrophic yeast Candida boidinii . Biosci Biotechnol Biochem , 2009, 73(4): 793-800.
[95]
Machida M, Yamada O, Gomi K. Genomics of Aspergillus oryzae : learning from the history of Koji mold and exploration of its future. DNA Res , 2008, 15(4): 173-183.
[96]
Stefanova G, Vlahova M, Atanassov A. Production of recombinant human lactoferrin from transgenic plants. Biol Plant , 2008, 52(3): 423-428.
[97]
Zhang Z, Coyne DP, Vidaver AK, Mitra A. Expression of human lactoferrinc DNA confers resistance to Ralstoniasola nacearum in transgenic tobacco plants. Phytopathology , 1998, 88(7): 730-734.
[98]
Humphrey BD, Huang N, Klasing KC. Rice expressing lactoferrin and lysozyme has antibiotic-like properties when fed to chicks. J Nutr , 2002, 132(6): 1214-1218.
[99]
He Y, Ning TT, Xie TT, Qiu QC, Zhang LP, Sun YF, Jiang DM, Fu K, Yin F, Zhang WJ, Shen L, Wang H, Li JJ, Lin QS, Sun YX, Li HZ, Zhu YG, Yang DC. Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Natl Acad Sci USA , 2011, 108(47): 19078-19083.
[100]
Suzuki YA, Kelleher SL, Yalda D, Wu LY, Huang JM, Huang N, Lönnerdal B. Expression, characterization, and biologic activity of recombinant human lactoferrin in rice. J Pediatr Gastroenterol Nutr , 2003, 36(2): 190-199.
[101]
Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM. Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes. Nature , 1982, 300(5893): 611-615.
[102]
Appel MJ, van Veen HA, Vietsch H, Salaheddine M, Nuijens JH, Ziere B, de Loos F. Sub-chronic (13-week) oral toxicity study in rats with recombinant human lactoferrin produced in the milk of transgenic cows. Food Chem Toxicol , 2006, 44(7): 964-973.
[103]
Hu WP, Zhao J, Wang JW, Yu T, Wang J, Li N. Transgenic milk containing recombinant human lactoferrin modulates the intestinal flora in piglets. Biochem Cell Biol , 2012, 90(3): 485-496.
[3]
Lönnerdal B, Iyer S. Lactoferrin: molecular structure and biological function. Annu Rev Nutr , 1995, 15(1): 93-110.
Chen K, Zhang L, Li H, Zhang Y, Xie H, Shang J, Tian WZ, Yang P, Chai LY, Mao M. Iron metabolism in infants: influence of bovine lactoferrin from iron-fortified formula. Nutrition , 2015, 31(2): 304-309.
[6]
Valenti P, Berlutti F, Conte MP, Longhi C, Seganti L. Lactoferrin functions: current status and perspectives. J Clin Gastroenterol , 2004, 38(Suppl. 6): S127-S129.
[7]
Wakabayashi H, Oda H, Yamauchi K, Abe F. Lactoferrin for prevention of common viral infections. J Infect Chemother , 2014, 20(11): 666-671.
[8]
van der Strate BW, Beljaars L, Molema G, Harmsen MC, Meijer DK. Antiviral activities of lactoferrin. Antiviral Res , 2001, 52(3): 225-239.
[9]
Zhang YL, Lima CF, Rodrigues LR. In vitro evaluation of bovine lactoferrin potential as an anticancer agent. Int Dairy J , 2015, 40: 6-15.
[10]
de la Rosa G, Yang D, Tewary P, Varadhachary A, Oppenheim JJ. Lactoferrin acts as an alarmin to promote the recruitment and activation of APCs and antigen-specific immune responses. J Immunol , 2008, 180(10): 6868-6876.
[11]
Kruzel ML, Actor JK, Zimecki M, Wise J, Ploszaj P, Mirza S, Kruzel M, Hwang SA, Ba X, Boldogh I. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression. J Biotechnol , 2013, 168(4): 666-675.
[12]
Blais A, Fan CB, Voisin T, Aattouri N, Dubarry M, Blachier F, Tomé D. Effects of lactoferrin on intestinal epithelial cell growth and differentiation: an in vivo and in vitro study. Biometals , 2014, 27(5): 857-874.
[13]
Umuhumuza LC, Niu WM, Sun XL. Effect of bovine lactoferrin and casein peptide powder on microbial growth and glucose utilization by microorganisms in pork meat during storage at 4ºC. Pakistan J Nutrit , 2011, 10(3): 208-213.
[14]
Tamura Y. Production and application of bovine lactoferrin. Bull-Int Dairy Federat , 2004, (389): 64-68.
[15]
Balcão VM, Costa CI, Matos CM, Moutinho CG, Amorim M, Pintado ME, Gomes AP, Vila MM, Teixeira JA. Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applications. Food Hydrocolloids , 2013, 32(2): 425-431.
[16]
Metz-Boutigue MH, Jollès J, Mazurier J, Schoentgen F, Legrand D, Spik G, Montreuil J, Jollès P. Human lactotransferrin: amino acid sequence and structural comparisons with other transferrins. Eur J Biochem , 1984, 145(3): 659-676.
[17]
Bezault J, Bhimani R, Wiprovnick J, Furmanski P. Human lactoferrin inhibits growth of solid tumors and development of experimental metastases in mice. Cancer Res , 1994, 54(9): 2310-2312.
[18]
Byrne SL, Krishnamurthy D, Wessling-Resnick M. Pharmacology of iron transport. Annu Rev Pharmacol Toxicol , 2013, 53: 17-36.
[19]
Weinberg ED. Human lactoferrin: a novel therapeutic with broad spectrum potential. J Pharm Pharmacol , 2001, 53(10): 1303-1310.
[20]
Lambert LA, Perri H, Halbrooks PJ, Mason AB. Evolution of the transferrin family: conservation of residues associated with iron and anion binding. Comp Biochem Physiol B Biochem Mol Biol , 2005, 142(2): 129-141.
[21]
Sharma S, Sinha M, Kaushik S, Kaur P, Singh TP. C-lobe of lactoferrin: the whole story of the half-molecule. Biochem Res Int , 2013, 2013: Article ID 271641.
[22]
Wang JR, Tian ZG, Teng D, Yang YL, Hu JC, Wang JH. Cloning, expression and characterization of Kunming mice lactoferrin and its N-lobe. Biometals , 2010, 23(3): 523-530.
[23]
Johansson B. Chromatographic separation of lactalbumin from human milk whey on calcium phosphate columns. Nature , 1958, 181(4614): 996-997.
[24]
Nagasako Y, Saito H, Tamura Y, Shimamura S, Tomita M. Iron-binding properties of bovine lactoferrin in iron-rich solution. J Dairy Sci , 1993, 76(7): 1876-1881.
[25]
Dionysius DA, Grieve PA, Milne JM. Forms of lactoferrin: their antibacterial effect on enterotoxigenic Escherichia coli . J Dairy Sci , 1993, 76(9): 2597-2600.
[26]
Baker HM, Anderson BF, Baker EN. Dealing with iron: common structural principles in proteins that transport iron and heme. Proc Natl Acad Sci USA , 2003, 100(7): 3579-3583.
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