Identification, Evolution, and Regulation of Expression of Guinea Pig Trappin with an Unusually Long Transglutaminase Substrate Domain

Soichi Kojima,Shigehisa Hirose,Ryoji Kawai,Taku Hirata,Akira Kato,Yutaka Furutani,In-Gyu Kim,Azzania Fibriani,Ju-Hong Jeon,Yasuhisa Fujii

doi:10.1074/jbc.m501678200

Abstract

Trappins are found in human, bovine, hippopotamus, and members of the pig family, but not in rat and mouse. To clarify the evolution of the trappin genes and the functional significance of their products, we isolated the trappin gene in guinea pig, a species belonging to a rodent family distinct from rat and mouse. Guinea pig trappin was confirmed to encode the same domain structure as trappin, consisting of a signal sequence, an extra large transglutaminase substrate domain, and a whey acidic protein motif. Northern blot analysis and in situ hybridization histochemistry as well as immunohistochemistry demonstrated that guinea pig trappin is expressed solely in the secretory epithelium of the seminal vesicle and that its expression is androgen-dependent. We confirmed that guinea pig trappin is cross-linked by prostate transglutaminase and that the whey acidic protein motif derived from guinea pig trappin has an inhibitory activity against leukocyte elastase. Genome sequence analysis showed that guinea pig trappin belongs to the family of REST (rapidly evolving seminal vesicle transcribed) genes.

Highlights

Trappins are a family of secreted proteins composed of two domains: a transglutaminase substrate (TGS)1 domain and a
Cloning and sequencing of its full-length cDNA indicated that caltrin II consists of only the whey acidic protein (WAP) motif and lacks the TGS domain [28]
We screened, using hippopotamus trappin cDNA as a probe, the guinea pig seminal vesicle cDNA library that had been constructed for the isolation of caltrin II cDNA and obtained several positive clones, including 12 clones corresponding to nucleotides 3184 –5087, 3504 –5070, 4054 –5087, 4315–5087, 4432– 5087, and 4452–5087 of guinea pig trappin cDNA (Fig. 1D)

Summary

EXPERIMENTAL PROCEDURES

Materials—Guinea pigs (male, 7 weeks old) were obtained from Tokyo Laboratory Animal Science Co., Ltd. (Tokyo, Japan). Digoxigenin-labeled RNA probes were synthesized from guinea pig trappin cDNA using T3 or T7 RNA polymerase, and digoxigeninlabeled nucleotides were prepared using a digoxigenin RNA labeling kit (Roche Applied Science) according to the manufacturer’s instructions. The supernatants were checked by SDS-PAGE under nonreducing conditions, and the concentrations of recombinant guinea pig trappin were determined using the BCA protein assay kit. R-Tgs-Gpt (1 ␮g) prepared as described above was incubated with 0.5 ␮g of His6-Xpress-GFP and 0.31 ␮g of guinea pig liver transglutaminase (Oriental Yeast Co., Tokyo) in transglutaminase assay buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, and 5 mM CaCl2 in the presence and absence of 50 mM EDTA, a transglutaminase inhibitor. Dixon analysis was used for determining the type of inhibition and for calculating Ki values [39]

RESULTS

DISCUSSION

Pancreatic elastase Ki