Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)

Abstract

Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.

Figure 1

Alignment of spiggin amino acid sequences. A. Conservation of the N-terminal region containing the signal peptide (boxed), the sequences utilized to synthesize the KTK and HRD peptides (bold with double line above sequence) and the glycosylation sequence (bold with single line above sequence). B. The central region of all spiggin proteins, except spiggin γ, contains one polymerization site (bold with single line above sequence). C. The C-terminal region of Spiggin 1.1 and 4, contain one additional polymerization site (bold with single line above sequence). Spaces (-) are inserted to allow the alignment of homologous amino acids.

Figure 2

Representative titration curves of antibody dilution curves. For the peptide antibodies the curve was produced against known amounts of peptide while the native antibody was titrated against native spiggin. Each dilution was measured in triplicate and a relative value was calculated from the mean values of the lowest dilution.

Figure 3

Determination of linear detection range. Serial dilutions of the HRD peptide in conjunction with HRD antibody diluted 1: 5000, HRD peptide in conjunction with affinity purified HRD antibody diluted 1: 10000, and KTK peptide in conjunction with KTK antibody diluted 1: 5000. Each dilution was measured in triplicate.

Figure 4

A. Western blot analysis of kidney and urinary bladder content. A. Kidney cytosol from male three-spined stickleback was used to determine the presence of spiggin isoforms using antibodies against KTK and HRD respectively. B. Western blot analysis of spiggin content of male and female three-spined stickleback kidney and urinary bladder using antibodies directed against native spiggin. The deduced molecular masses of different spiggin isoforms are indicated on the right. Ki = Kidney, UB = Urinary bladder. Molecular mass of the protein standard is indicated on the left (kDa).

Figure 5

Quantification of renal spiggin levels from non-spawning males and females and pre-spawning males using the HRD antibody. A. Spiggin content in nmol per kidney. B. Spiggin levels in nmol per mg kidney.

Figure 6

Correlation of spiggin levels to kidney somatic index and body weight. A. The total spiggin content (nmol) showed strong correlated to KSI. (p < 0.0001, r² = 0.95) B. The total spiggin content (nmol) showed no correlation to BW.

Figure 7

Spiggin induction in 11-KT exposed fish. Three-spined sticklebacks were exposed to 5 μg 11-KT/kg BW for three days. Renal spiggin levels were measured using the HRD antibody. Five animals were used for the control group while 4 animals were used for the exposed group. *, Statistically significant difference from the control (p < 0.05).

Figure 8

Spiggin γ expression in cell and tissue lysates was examined by Western blot. A. Detection of recombinant spiggin γ protein using the polyclonal native spiggin antibody; B. detection of His-tagged proteins using anti-His-tag antibody. MW – molecular weight markers (kDa); Control – bacterial lysate prior to IPTG addition; Expressed – bacterial supernatant following IPTG addition. The arrows indicate the position of spiggin γ protein.