Unlike mammalian GRIFIN, the zebrafish homologue (DrGRIFIN) represents a functional carbohydrate-binding galectin

Abstract

Galectins, a family of beta-galactoside-binding proteins, participate in a variety of biological processes, such as early development, tissue organization, immune regulation, and tumor evasion and metastasis. Although as many as fifteen bona fide galectins have been identified in mammals, but the detailed mechanisms of their biological roles still remain unclear for most. This fragmentary knowledge extends to galectin-like proteins such as the rat lens crystallin protein GRIFIN (Galectin-related inter fiber protein) and the galectin-related protein GRP (previously HSPC159; hematopoietic stem cell precursor) that lack carbohydrate-binding activity. Their inclusion in the galectin family has been debated, as they are considered products of evolutionary co-option. We have identified a homologue of the GRIFIN in zebrafish (Danio rerio) (designated DrGRIFIN), which like the mammalian equivalent is expressed in the lens, particularly in the fiber cells, as revealed by whole mount in situ hybridization and immunostaining of 2 dpf (days post fertilization) embryos. As evidenced by RT-PCR, it is weakly expressed in the embryos as early as 21 hpf (hour post fertilization) but strongly at all later stages tested (30 hpf and 3, 4, 6, and 7 dpf). In adult zebrafish tissues, however, DrGRIFIN is also expressed in oocytes, brain, and intestine. Unlike the mammalian homologue, DrGRIFIN contains all amino acids critical for binding to carbohydrate ligands and its activity was confirmed as the recombinant DrGRIFIN could be purified to homogeneity by affinity chromatography on a lactosyl-Sepharose column. Therefore, DrGRIFIN is a bona fide galectin family member that in addition to its carbohydrate-binding properties, may also function as a crystallin.

Fig. 1

(A) Homology modeling of DrGRIFIN. Ribbon diagram showing the overlap of the DrGRIFIN (green) and the bovine gal1 structure (white, PDB code 1SLT, ref. 9). The amino acids that are essential for carbohydrate-binding are shown in green for DrGRIFIN, and yellow for bovine gal1. (B) Genomic organization of DrGRIFIN. The vertical boxes represent exons, which are numbered at the top. The size of each exon (in bp) is indicated at the bottom. The horizontal boxes represent introns, whose sizes are indicated in kb in the middle.

Fig. 2

(A) Purification of recombinant DrGRIFIN on a lactosyl-Sepharose. The extract was absorbed on lactosyl-Sepahrose, and the bound protein was eluted with 0.1 M glucose followed by 0.1 M lactose. Samples of each step were electrophoresed and silver-stained. (B). Serological cross-reactivity of anti-rat GRIFIN antibody with DrGRIFIN.

Fig. 3

Expression of DrGRIFIN in zebrafish identified by RT-PCR. (A) Temporal expression patterns; (B) Spatial expression patterns (tissue/organ-specific) in the adult C-G. Whole mount in situ hybridization and immunostaining. (C, D) In situ hybridization of whole zebrafish embryo (2 days post fertilization, dpf) showing (C) DrGRIFIN expression in lens (lateral view); (D) 2 dpf embryos (lateral view) with sense probe for negative control. E, F. Whole mount immunostaining with anti-rat GRIFIN monoclonal antibody showing DrGRIFIN expression in lens. E and F are the same, but with different time of substrate development. G. Cross-section of eye after whole mount in situ hybridization.