Tgm1-like transglutaminases in tilapia (Oreochromis mossambicus)

Abstract

Among the adaptations of aquatic species during evolution of terrestrial tetrapods was the development of an epidermis preventing desiccation. In present day mammals, keratinocytes of the epidermis, using a membrane-bound transglutaminase (Tgm1), accomplish this function by synthesizing a scaffold of cross-linked protein to which a lipid envelope is attached. This study characterizes the abilities of two homologous transglutaminase isozymes in the teleost fish tilapia to form cross-linked protein structures and their expression in certain tissues. Results indicate they are capable of membrane localization and of generating cellular structures resistant to detergent solubilization. They are both expressed in epithelial cells of the lip, buccal cavity and tips of gill filaments. Adaptation of transglutaminase use in evolution of terrestrial keratinocytes evidently involved refinements in tissue expression, access to suitable substrate proteins and activation of cross-linking during terminal differentiation.

Fig 1. Cladogram of O. niloticus transglutaminase sequences.

Genbank was searched using BLASTP for the active site motif “GQCWVF”. Sequences are identified in the left margin by number: 1, 2, 3, 5, 13 (XIIIa) and NA (not annotated); L, like; lower case letters arbitrarily distinguish those of the same type. The sequences identified as transglutaminases were aligned using the Clustal Omega algorithm with default parameters (available at http://www.ebi.ac.uk/Tools/services/web/toolresult.ebi?jobId=clustalo-I20160718-224602-0849-78264976-oy&analysis=phylotree) to generate the cladogram showing relative estimated evolutionary distances. A red box shows the two Tgm1-like (1La and 1Lb) sequences. The sequence with an inactivating cysteine to serine substitution in the active site (Genbank accession XP_005461247.1) was not included. Entries 5Lb and 5Lc differ by the former having a single extra amino acid, and thus they may not be distinct. Genbank accession numbers are given next to the Tgm.

Fig 2. Relative levels of Tgm1A and Tgm1B mRNA in cultured lip cells.

Values are presented for 3 independent samples (mean ± std dev) relative to those of the housekeeping gene, Talin1.

Fig 3. Stimulation of envelope cross-linking by the ionophore X537A.

Shown is the degree of light scattering (A³⁴⁰) by cross-linked envelopes isolated after addition of SDS and DTT. (A) Confluent cultures of OmL cells with (X537A) and without (Con) overnight treatment with ionophore; compilation of 4 independent experiments. (B) HEK293FT cells not transfected (Con) or transfected with the full length coding region of Tgm1A or Tgm1B; two days after transfection, cultures were treated overnight with ionophore; compilation of two independent experiments. In each panel, differences between treated and control samples were judged significant (p<0.01) by ANOVA using STATA SE9 statistical software.

Fig 4. Envelopes from cultured lip and HEK293 cells, all treated with SDS and DTT.

Confluent OmL cells were treated overnight with X537A (A) or harvested after spontaneous detachment (B). HEK293FT cells were transfected with Tgm1A (C) or Tgm1B (D) full length coding regions for two days and then overnight with X537A ionophore.

Fig 5. Membrane anchorage of Tgm1A and Tgm1B.

(A) Extracts from cultured lip cells showed immunoreactive bands with the expected mobilities of 86 kDa (784 and 786 amino acids, respectively) in both particulate (P) and soluble (S) fractions.(B) Extracts from HEK293 cells also showed substantial immunoreactive bands in particulate and soluble fractions when transfected with cDNA encoding the wild type coding region (a). Transfections with constructs where the cysteine cluster (CPCCC) was deleted (b) or was replaced by AIAAA (c) exhibited little if any Tgm1 in the particulate fraction.

Fig 6. Sections of lip.

(A) Hematoxylin and eosin stained. Thin arrow points to epithelium with nucleated cells; thick arrow points to a wide layer of connective tissue with abundant eosin-positive collagen. (B) Immunostained section without primary antibody (negative control). (C) The thin arrow points to Tgm1A immunoreactivity (brown) seen throughout the epithelium, although not uniformly. Some nests of cells, primarily in the lower half of the epithelium and connected to the basal layer, showed lower immunoreactivity than elsewhere (example shown by thick arrow on the right). (D) Tgm1B immunoreactivity (brown) exhibited the same pattern as Tgm1A. The thin arrow points to the brown-stained epithelium, and the thick arrow at right shows a region of lower stain. Each section was counterstained with hematoxylin. Scale bar = 100 μm.

Fig 7. Sections of buccal epithelium.

(A) Hematoxylin and eosin stained. Thin arrow points to the stratified epithelium with nucleated cells; thick arrow points to eosin-positive collagen at the border with underlying connective tissue. (B) Immunostained section without primary antibody (negative control). (C) Tgm1A immunoreactivity. The thick arrow points to most concentrated staining of cells at the surface of the epithelium, and the thin arrow points to a glandular cell at the surface releasing mucus. (D) Tgm1B immunoreactivity. Arrows as in C. (E) Periodic acid-Schiff stain showing high density of mucus cells (thin arrow). Thick arrow points to collagen at interface with connective tissue. (F) negative control for section E. Each section was counterstained with hematoxylin. Scale bar = 100 μm.

Fig 8. Sections of gill filaments.

(A) Low magnification of filament stained with hematoxylin and eosin. Features include the stratified epithelium at the tip of the filament (1), sinus venosus (2), sinus with erythrocytes (3), intralamellar filament epithelium (4) and secondary lamella (5). (B) Hematoxylin and eosin-stained section at higher magnification showing features 1–4 in section A. (C) Immunostained section without primary antibody (negative control); (D) Tgm1A immunoreactivity. Arrow points to most intense staining at the tip. (E) Tgm1B immunoreactivity. Similar to section D, the arrow points to the most intense staining at the tip. Each section was counterstained with hematoxylin. Scale bar = 34 μm (A) and 50 μm (B-E).