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Measurement of Betacellulin Levels in Bovine Serum, Colostrum and Milk

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Measurement of Betacellulin Levels in Bovine Serum, Colostrum and Milk 203 Measurement of betacellulin levels in bovine serum, colostrum and milk S E P Bastian, A J Dunbar, I K Priebe, P C Owens1 and C Goddard2 Cooperative Research Centre for Tissue Growth and Repair, CSIRO Health Sciences and Nutrition, PO Box 10041, Adelaide BC, SA 5000, Australia 1Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide, SA 5005, Australia 2GroPep Limited, 28 Dalgleish Street, Thebarton, SA 5031, Australia (Requests for offprints should be addressed toSEPBastian, Cooperative Research Centre for Tissue Growth and Repair, PO Box 10041, Adelaide BC, SA 5000, Australia; Email: [email protected]) Abstract Betacellulin, a member of the epidermal growth factor exceeded that of betacellulin in serum from male calves 1 (EGF) family, was originally isolated and identified from and 5 weeks of age (0·530·15 ng/ml and 0·70 the conditioned medium from a murine pancreatic -cell 0·09 ng/ml respectively; mean...; n=9). Betacellu- carcinoma cell line. Recently, we isolated bovine betacel- lin measured in the serum of these same animals when lulin from a growth factor enriched cheese whey extract, aged between 27 and 43 weeks was below the detection but there is no information on the presence of betacellulin limits of the RIA. Sera from 10 out of 36 unmated heifers in other biological fluids. We have cloned the cDNA for contained betacellulin levels within the detection limits of bovine betacellulin, produced recombinant betacellulin the assay (0·4330·06 ng/ml mean...; n=10). The and shown that it has a similar potency to the purified presence of betacellulin in bovine colostrum and milk native molecule in stimulating the proliferation of Balb/ suggests that it plays a role in the growth and development c3T3 fibroblasts. We have produced a polyclonal anti- of the neonate and/or mammary gland function. The serum to bovine betacellulin which did not cross-react results also show that betacellulin is undetectable in the with EGF or transforming growth factor- (TGF-). The castrated adult male circulation. Additionally, although antibody was used in a homologous RIA that was able to present in very low amounts, serum betacellulin could be detect betacellulin in pooled bovine colostrum sampled under hormonal regulation in the female, since betacellu- during the first 3 days after calving (2·300·11 ng/ml lin was detected in sera from 27% of the unmated heifers mean...; n=6), in bovine milk soluble fraction examined in this study. The high levels of betacellulin (1·930·64 ng/ml mean...; n=5) and in bovine detected in FBS relative to newborn and adult serum cheese whey (2·590·16 ng/ml mean...; n=3). suggests a possible endocrine role for this growth factor in The betacellulin concentration in foetal bovine serum the bovine foetus. (FBS) (3·680·59 ng/ml mean...; n=6) greatly Journal of Endocrinology (2001) 168, 203–212 Introduction transforming growth factor- (Marchionni et al. 1993, Carraway et al. 1997, Chang et al. 1997, Higashiyama et al. Betacellulin was first isolated as a mitogen in the con- 1997, Raab & Klagsbrun 1997, Zhang et al. 1997). ditioned medium of a mouse pancreatic -cell carcinoma Betacellulin shares significant sequence homology with the cell line (Shing et al. 1993). Mouse betacellulin is a 32 kDa other members, including a characteristic six-cysteine glycosylated polypeptide composed of 80 amino acids consensus motif within the EGF-like domain, which forms which shares 82·5% and 79% amino acid sequence identity three intra-molecular disulphide bonds. Another common with the human and bovine form respectively (Shing et al. feature of the EGF family members is that they are 1993, Watanabe et al. 1994, Dunbar et al. 1999). Betacel- produced as transmembrane precursor molecules and are lulin is one of the epidermal growth factor (EGF) family of proteolytically cleaved to give rise to the soluble mature growth factors. The other members include amphiregulin, growth factor. Betacellulin is able to bind and activate epiregulin, heparin-binding EGF-like growth factor members of the EGF tyrosine kinase receptor family (HB-EGF), neural- and thymus-derived activator for erbB encoded by the erbB genes, specifically, erbB-1 (EGFR) kinases (NTAK), the neuregulin gene products and and erbB-4 (Watanabe et al. 1994, Riese et al. 1996). It has Journal of Endocrinology (2001) 168, 203–212 Online version via http://www.endocrinology.org 0022–0795/01/0168–203 2001 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/27/2021 02:07:16PM via free access 204 S E P BASTIAN and others · Betacellulin in biological fluids since been demonstrated that betacellulin is not only able The cDNA encoding the complete nucleotide sequence to activate homodimers of erbB-1 and erbB-4, but also of mature bovine betacellulin [Asp1-Tyr80] was generated combinations of erbB receptor heterodimers, including the by RT-PCR. Total RNA was isolated from 80–90% oncogenic erbB-2–erbB-3 complex (Alimandi et al. 1997). confluent Madin Darby bovine kidney cells (MDBK, Betacellulin is expressed predominantly in pancreas and ATCC CCL 22) and cDNA synthesized from 1 µg total small intestine but also weakly in heart, lung, liver, skeletal MDBK RNA using oligo dT primer and Superscript II muscle, kidney, prostate, testis, ovary and colon (Sasada (Life Technologies, Melbourne, Australia). The subse- et al. 1993, Seno et al. 1996). It is a known mitogen for quent cDNA was used as a template for PCR with numerous cell types including smooth muscle cells, Balb/ oligonucleotide primers, 5 ATC TAG GTT ACC ATG c3T3 cells, Madin Darby canine kidney epithelial cells and GAT GGG AAT TCA ACC AGA 3 and 5 CTA GAT retinal pigment epithelial cells (Shing et al. 1993). Despite AAG CTT TCA TCA GTA AAA CAA GTC AAC this knowledge the biological role of betacellulin in vivo is TCT 3. The resultant 273 bp PCR product was puri- not yet understood. fied, digested with HpaI and HindIII and cloned into Bovine milk is rich in a number of growth factors the HpaI/HindIII digested expression vector p[Met1]- including platelet-derived growth factor (PDGF) and the pGH(1–46). The recombinant plasmid was maintained in insulin-like growth factors, IGF-I and IGF-II (Shing & E. coli, strain JM101. Klagsbrun 1984, Francis et al. 1988). Previous studies in The E. coli JM101 strain harbouring the plas- this laboratory have produced a novel whey extract that mid p[Met1]-pGH(1–46)-Val-Asn-Met-betacellulin was comprises a concentrated mixture of bovine milk proteins selected as a single colony and used to inoculate a 20 ml enriched in growth factors (Francis et al. 1995). This starter culture in a medium (K2HPO4 60 mM, KH2PO4 extract is a source of potent growth-promoting activity for 33 mM, (NH4)2SO4 7·5 mM, sodium citrate 1·7 mM, a number of mesoderm-derived cell types (Belford et al. MgSO4.7H2O 10 µM, -glucose 0·2%, thiamine 1995). A recent study by Dunbar et al. (1999) has since 0·0005%, ampicillin 50 µg/ml). The culture was grown at found that betacellulin accounts for about 50% of the 37 C for 16 h. The starter culture was in turn used to growth-promoting activity within this extract when tested inoculate two 5 l fermenters (Applikon, Schiedam, The in a Balb/3T3 bioassay. Netherlands) each containing 3 l of growth medium The implication of this finding is that betacellulin is (NH4Cl 30 mM, K2SO4 7 mM, KH2PO4 12 mM, present in bovine milk. Taken together with its role as a Na2HPO4 19 mM, -glucose 139 mM, MgSO4.7H2O mitogenic factor for a number of cell types and its 2·4 mM, thiamine 0·0004%, Fe2SO4.7H2O 35 µM, expression, along with its receptors in numerous tissues, MnSO4.7H2O7·4 µM, CuSO4.7H2O0·8 µM, tri- suggests a major biological role. Receptors for betacellulin sodium citrate 74 µM and ampicillin 50 µg/ml). Bacteria have been demonstrated in liver and placental tissue were grown at 37 C until the absorbance at 600 nm (O’Keefe et al. 1974, Wang et al. 1992, Mielke et al. reached an OD of 4·0 and then induced with IPTG 1998), which raises the possibility that betacellulin, may be (0·33 mM) and the cultivation was continued until glucose in the circulation. Using a homologous RIA for bovine became limiting, indicated by a sharp rise in pH. Regu- betacellulin, we were able to confirm the presence and lation of temperature, pH and oxygen was under auto- determine the levels of betacellulin in bovine colostrum matic control (FC4 Data system, Real Time Engineering, and milk. Additionally, we demonstrated the presence of Sydney, Australia). Cells were disrupted at 5000 p.s.i. betacellulin in serum, and the effects of age and sex of the following five passes through a homogenizer (Rannie animal on the serum betacellulin concentration. Instruments, APV Homogenisers, Albertslund, Denmark) and inclusion bodies collected by centrifugation Materials and Methods (10 000 r.p.m., 25 min, 4 C). The inclusion bodies were washed twice with NaCl (30 mM), KH PO (10 mM), Production of recombinant bovine betacellulin 2 4 ZnCl2 (0·5 mM), harvested by centrifugation at Amodification of the expression vector p[Met1]-pGH 6000 r.p.m. and stored at80 C. (1–46)IGF-I (King et al. 1992) was used to produce Washed inclusion bodies, in 20 g batches, were thawed, recombinant bovine betacellulin. This plasmid directs suspended at 10% (w/v) in a buffer containing urea (8 M), isopropyl--galactoside (IPTG)-induced high-level ex- Tris–HCl (0·1 M), glycine (40 mM), dithiothreitol pression of betacellulin as a fusion protein that has a (40 mM) and ZnCl2 (0·5 mM) (pH 9·0) and stirred for 46 amino acid amino-terminal extension derived from 30 min at room temperature.
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