Insulin-like growth factor binding protein 2 is a growth inhibitory protein conserved in zebrafish

Abstract

Fish serum contains several specific binding proteins for insulin-like growth factors (IGFBPs). The structure and physiological function of these fish IGFBPs are unknown. Here we report the complete primary sequence of a zebrafish IGFBP deduced from cDNA clones isolated by library screening and rapid amplification of cDNA ends. The full-length 1,757-bp cDNA encodes a protein of 276 aa, which contains a putative 22-residue signal peptide and a 254-residue mature protein. The mature zebrafish IGFBP has a predicted molecular size of 28,440 Da and shows high sequence identity with human IGFBP-2 (52%). The sequence identities with other human IGFBPs are <37%. Chinese hamster ovary cells stably transfected with the zebrafish IGFBP-2 cDNA secreted a 31-kDa protein, which bound to IGF-I and IGF-II with high affinity, but did not bind to Des(1-3)IGF-I or insulin. Northern blot analyses revealed that the zebrafish IGFBP-2 transcript is a 1.8-kb band expressed in many embryonic and adult tissues. In adult zebrafish, IGFBP-2 mRNA levels were greatly reduced by growth hormone treatment but increased by prolonged fasting. When overexpressed or added to cultured zebrafish and mammalian cells, the zebrafish IGFBP-2 significantly inhibited IGF-I-stimulated cell proliferation and DNA synthesis. These results indicate that zebrafish IGFBP-2 is a negative growth regulator acting downstream in the growth hormone-IGF-I axis.

Figure 1

Nucleotide and deduced amino acid sequence of the zebrafish IGFBP-2. The first amino acid residue of the predicted mature protein is indicated by *. The GenBank database accession no. is AF198033.

Figure 2

Comparison of the zebrafish IGFBP-2 primary sequence with that of human (27), bovine (37), ovine (38), mouse (39), rat (29), and chicken (40). Sequence alignment was obtained with macvector by the Clustal method. Gaps were introduced to maximize sequence homologies. The first amino acid residue of the predicted mature protein is indicated by *. Residues that are identical or conserved are shaded. The consensus sequence is shown on the bottom line.

Figure 3

The zebrafish IGFBP-2 cDNA encodes a protein that binds to IGFs but not to insulin and Des(1–3)IGF-I. (A) Ligand blot analysis of CM prepared from the pRc/CMV-IGFBP-2-transfected clones 2, 5, and 6 (lanes 2–4), the pRC/CMV vector-transfected clones 1 and 2 (lanes 5 and 6), and pure bovine IGFBP-2 (lane 1). (B) Affinity crosslinking of [¹²⁵I] IGF-I to zebrafish IGFBP-2 produced by CHO cells (IGFBP-2-transfected clone 2). Binding was competed by the addition of no competitor (lane 2), 500 ng of IGF-I (lane 3), IGF-II (lane 4), Des(1–3)IGF-I (lane 5), and insulin (lane 6). Lane 1 is the negative control that did not have any zebrafish IGFBP-2. The competition of [¹²⁵I] IGF-I was similar in all other IGFBP-2-transfected clones examined (clones 5 and 6). (C) Affinity crosslinking of [¹²⁵I] IGF-II to zebrafish IGFBP-2 produced by CHO cells (IGFBP-2-transfected clone 2). Binding was competed by the addition of no competitor (lane 2), 500 ng of IGF-I (lane 3), IGF-II (lane 4), Des(1–3)IGF-I (lane 5), and insulin (lane 6). Lane 1 is the negative control that did not have any zebrafish IGFBP-2. The competition of [¹²⁵I] human IGF-II was similar in other IGFBP-2-transfected clones examined (clones 5 and 6).

Figure 4

The expression of IGFBP-2 gene is increased by fasting and decreased by GH treatment in zebrafish. (A) Tissue distribution pattern of IGFBP-2 mRNA. Northern blot analysis of RNA samples isolated from 20-hr embryos (lane 1), whole body of adult zebrafish (lane 2), and brain (lane 3), eye (lane 4), ovary (lane 5), liver (lane 6), intestine (lane 7), muscle (lane 8) and fin (lane 9) collected from adult fish. (B) Effect of fasting on IGFBP-2 mRNA expression. RNA was isolated from fed (control) or fasted adult zebrafish. The values are means ± SE of three animals. *, P < 0.05 compared with the control. (C) Effect of GH on IGFBP-2 mRNA expression. Adult zebrafish were injected with pure seabass GH (1 μg/ml) or saline and sacrificed 15, 24, or 48 hr after the injection. The values are means ± SE of three animals. *, P < 0.05 compared with the control.

Figure 5

Zebrafish IGFBP-2 inhibits cell growth and DNA synthesis. (A) Overexpression of zebrafish IGFBP-2 inhibits CHO cell growth. Growth curves of wild-type CHO cells (□), the pRC/CMV vector-transfected clone 1 (○) and clone 2 (▵), and the pRc/CMV-IGFBP-2-transfected clone 2 (●) and clone 6 (▴) are shown. *, P < 0.01 (n = 8) compared with the wild-type controls. (B) Effect of zebrafish IGFBP-2 on IGF-I-stimulated DNA synthesis in cultured zebrafish cells. Confluent cells were exposed to serum-free medium or IGF-I (100 ng/ml) in the presence of various concentrations of CM prepared from wild-type (open bars), mock-transfected (hatched bars), and zebrafish IGFBP-2-transfected cells (filled bars). The values are means ± SE of three separate experiments, each of which was performed in triplicate. *, P < 0.05 compared with the IGF-I alone group.