doi:10.1006/cyto.2001.0910, available online at http://www.idealibrary.com on

SHORT COMMUNICATION

ISOLATION, NUCLEOTIDE SEQUENCE ANDEXPRESSION OF A cDNA ENCODING FELINE

GRANULOCYTE COLONY-STIMULATINGFACTOR

Stephen P. Dunham and David E. Onions

A cDNA encoding feline granulocyte colony stimulating factor (fG-CSF) was cloned fromalveolar macrophages using the reverse transcriptase-polymerase chain reaction. The cDNA is949 bp in length and encodes a predicted mature protein of 174 amino acids. RecombinantfG-CSF was expressed as a glutathione S-transferase fusion and purified by affinity chroma-tography. Biological activity of the recombinant protein was demonstrated using the murinemyeloblastic cell line GNFS-60, which showed an ED50 for fG-CSF of approximately 2 ng/ml.

� 2001 Academic Press

Granulocyte colony-stimulating factor (G-CSF orCSF-3) belongs to a family of colony-stimulatingfactors required for the survival, proliferation anddifferentiation of haemopoietic progenitor cellsand acts principally upon mature progenitors of theneutrophil lineage.1 G-CSF is a key cytokine in theregulation of host immunity and clinical applicationsfor the recombinant cytokine have been widelyexplored.2 Patients with life threatening neutropeniaare typical candidates for treatment with recombinanthuman G-CSF (rhG-CSF), the use of which can reduceinfections and hospital admissions.

The cDNAs for G-CSF have been cloned from anumber of species including: man,3 mice,4 rat,5 pig,6

cattle7 and sheep.8 The cloning of the feline gene isdesirable because whilst the recombinant G-CSF fromother species is biologically active in cats, the homolo-gous cytokine may be more potent. Furthermore, theuse of rhG-CSF in cats is often associated with thedevelopment of neutralising antibodies.9 Feline G-CSF(fG-CSF) could be used to treat neutropenias in cats

CYTOKINE, Vol. 14, No. 6 (21 June), 2001: pp 347–351

such as those due to viral infections or myelosupressiondue to chemotherapy.

From the Department of Veterinary Pathology, University ofGlasgow, Bearsden Road, G61 1QH, UK

Correspondence to: S. P. Dunham, Retrovirus ResearchLaboratory, Department of Veterinary Pathology, University ofGlasgow, Bearsden Road, Glasgow. G61 1QH, UK. E-mail:s.dunham@vet.gla.ac.uk

Received 27 November 2000; accepted for publication 26 May 2001� 2001 Academic Press1043–4666/01/060347+05 $35.00/0

KEY WORDS: feline G-CSF/polymerase chain reaction/nucleotidesequence/cytokine/bacterial expression

RESULTS

The cloned fG-CSF cDNA is 949 bp in length andencodes a predicted mature protein of 174 amino acidsand part of the coding sequence for a secretory signalpeptide (Fig. 1). The consensus sequence is 85%identical to human, 76% to murine, 87% to ovine, 77%to rat, and 82% to bovine cDNAs. A recent databasesearch reveals an alternate cDNA sequence for fG-CSF(accession number AB042552). This is identical to thesequence reported here except for a single nucleotidechange at T237 (T to C) which does not alter thededuced protein sequence and minor differences at the5� and 3� ends of the sequence (because of the use ofdifferent primers for PCR amplification).

The predicted feline protein shows high homologyto other mammalian G-CSF proteins (Fig. 2). Likeother species, fG-CSF contains no potential N-linkedglycosylation sites. O-linked glycosylation of Thr133

results in greater stability of the human protein;10 thissite is conserved in fG-CSF. Two disulphide bridges,within hG-CSF are required for full biologicalactivity;10 the analogous cysteine residues are con-served between species (Fig. 2). Interestingly, the felinesequence AB042552 shows a signal peptide of 20 aminoacids, compared to 30 amino acids for human3 andmurine4 G-CSF.

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Recombinant fG-CSF was identified as a productof 22 kDa following thrombin cleavage of the gluta-thione S-transferase (GST) fusion protein (Fig. 3).Biological activity of fG-CSF was demonstrated usingthe murine myeloblastic cell line GNFS-60, whichproliferates in response to rhG-CSF.11 The prolifer-ation response curve for cells stimulated with eitherrecombinant feline or human G-CSF is shown inFig. 4. The logarithmic phase of these plots are paral-lel, confirming that the recombinant feline protein isanalagous to hG-CSF.12 The ED50 of GNFS-60 cellsfor hG-CSF is approximately 0.05 ng/ml, within theexpected limits of the assay (0.02–0.06 ng/ml). The cellsare approximately 100-fold less responsive to fG-CSF.

Figure 1. Nucleotide sequence and deduced amino acid sequence of feline granulocyte colony stimulating factor.

Oligonucleotide primers used in the PCR reaction are overlined. An asterisk indicates the position of the stop codon. The nucleotide sequencedata for feline G-CSF appears in the EMBL database with the accession number Y08558.

DISCUSSION

Feline G-CSF shows high homology to thecyokine in other species. However, the difference inbioactivity of fG-CSF compared to hG-CSF may beattributed to differences in key amino acids betweenthe cytokines. Residues critical for biological activityof human G-CSF13 are conserved in the feline proteinwith the exception of valine 124 which is substituted byleucine. Whilst mutagenesis of this site with a leucine toalanine substitution reduced hG-CSF activity to 41%of normal, the substitution of valine is relatively con-served and may have less effect. Other amino acidchanges within the feline protein compared to the

Feline G-CSF: cDNA sequence and expression / 349

human homologue may combine to produce themarked difference in activity on GNFS-60 cells. Inparticular, residues 20-58 have been implicated inreceptor binding.14 This region shows a number ofamino acid changes compared to the human or murineproteins. Despite their relatively low sensitivity,GNFS-60 cells are a convenient assay for fG-CSFwhich will aid the optimisation of protein expressionand purification.

The availability of recombinant fG-CSF will allowfurther evaluation of its activity in vitro. In particular,the use of feline bone marrow cells may provide a moreaccurate indication of its specific bioactivity. Theactivity of fG-CSF could be further determined in vivo,both in normal animals and those with natural andexperimental disease. An area of particular interestwithin our laboratory is the study of feline immuno-deficiency virus, an animal model for HIV. The use ofG-CSF in treatment of HIV infected patients has thepotential to increase viral replication within haemo-

poietic progenitor cells leading to a deleterious increasein viral loads. Availability of fG-CSF will permitstudies of this potential in the feline model.

MATERIALS AND METHODS

Animals and cellsAlveolar macrophages were isolated at post mortem by

bronchoalveolar lavage and cultured in Dulbecco’s modifiedEagles medium containing 20 mM HEPES, 2 mM glutamineand 10% FCS at 37�C, 5% CO2. After culture for 4 h,medium and non-adherent cells were removed, freshmedium added and the cells stimulated for 4 h with 10 �g/mllipopolysaccaride (Sigma, Poole, UK).

Figure 2. Comparison of the predicted amino acid sequence of feline G-CSF with human, murine, ovine, bovine and rat proteins.

Comparisons were made using the University of Wisconsin Genetics Computer Group program ‘BestFit’. The feline sequence is shown as theconsensus; dashes indicate complete identity with the feline sequence whilst a period indicates that the corresponding amino acid is absent.Conserved cysteine residues are indicated by asterisks. The position of the non-conserved residue Val124 is shown by an arrow (�). Numberingis in relation to the mature protein sequence. Comparison to the fG-CSF sequence AB042552 is also shown; this is identical other than for the5� end of the predicted protein (differences occurring due to the oligonucleotide primer used in the PCR of this feline sequence).

Cloning and sequencing of fG-CSFMessenger RNA was isolated from stimulated macro-

phages using an mRNA purification kit (Pharmacia Biotech,Bucks, UK). Complimentary DNA was synthesised from

350 / Dunham and Onions CYTOKINE, Vol. 14, No. 6 (21 June, 2001: 347–351)

NotI-d(T)18 primed mRNA using a First-strand cDNA syn-thesis kit (Pharmacia Biotech). Oligonucleotide primers usedfor PCR are shown in Figure 1. The reaction mix contained125 �M each dNTP, 1� PCR buffer, 2.5 units AmpliTaqDNA polymerase (Applied Biosystems, Warrington, UK)and 1.0 �M of each primer in 50 �l volume. Thermal cyclingconditions were: 94�C for 5 min; 30 cycles of 94�C for 1 min,60�C for 1 min and 72�C for 1 min; 72�C for 5 min. The PCR

product was cloned into pCRII (Invitrogen, Groningen,Netherlands) and sequenced using a Sequitherm long-readcycle-sequencing kit (Cambio, Cambridge, UK). Threeclones from each of two separate PCRs were sequenced onboth strands and a consensus sequence derived.

Expression of feline recombinant G-CSFThe coding region for the predicted mature fG-CSF

protein (nt 62 to 583) was amplified by PCR with Pfu DNApolymerase (Stratagene, Amsterdam, Netherlands) from aconsensus clone using the primers: 5�-GCGCGGATCCACCCCCTTGGGCCCTACCAG-3� (containing a BamHI site)and 5�-GCGCGGCCGCATTAGGGCTTGGTGAAGTGAAGTGG-3� (containing a NotI site). The amplified productwas subcloned into pGEX-4T-1 (Pharmacia Biotech) and itsnucleotide sequence verified by sequencing. The resultantplasmid was used to transfect BL21 E. coli. Transfected cellswere grown in 2YT medium at 30�C to an OD600 of 1.0.IPTG was added to 0.1 mM and cultures incubated at25�C for 120 min. The bacterial culture was pelleted andresuspended in ice cold PBS containing 1 mM PMSF. Cellswere lysed by sonication. Recombinant proteins were puri-fied by affinity chromatography using glutathione sepharose4B and the fusion protein cleaved by thrombin. The yield ofrecombinant protein was determined by Bradford assay(Bio-Rad Laboratories, Hemel Hempstead, UK) and purityestimated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis.

Measurement of fG-CSF bioactivityThe bioactivity of fG-CSF was measured using

GNFS-60 cells (with kind permission of M. Ono, ChugaiPharmaceuticals, Tokyo, Japan). The bioassay was per-formed as previously described15 using rhG-CSF (Filgrastim;a kind gift of Dr Mire-Sluis, NIBSC, UK) and purifiedpGEX protein as positive and negative controls respectively.

Acknowledgements

This work was funded by the Wellcome Trust andQ1 Biotech. Lesley Nicolson is thanked for usefuldiscussion regarding this manuscript.

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Figure 3. Coomassie stained SDS-PAGE of recombinant felineG-CSF proteins expressed in E. coli.

Lane 1. rfG-CSF following thrombin cleavage and affinity columnpurification (cleaved on column) Lane 2. rfG-CSF followingthrombin cleavage and affinity column purification (cleaved afterelution from column) Lane 3. GST protein. The likely identity offrG-CSF (in lanes 1 and 2), with an approximate size of 22 kDa, isindicated by an arrow. The GST protein has an approximate size of28 kDa and is also indicated; carry over of the GST moiety can beseen following affinity purification (lanes 1 and 2). The purity of therecombinant protein was estimated to be 10–20% and the yield20 �g/l of bacterial culture.

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Figure 4. Incorporation of 3H-thymidine by the murine cell lineGNFS-60 is increased by stimulation with feline G-CSF.

GNFS-60 cells respond to fG-CSF with an ED50 of approximately2 ng/ml. Minimal proliferation is seen in response to the GST proteinalone and only at higher protein concentrations.

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