In situ detection of active transglutaminases for keratinocyte type (TGase 1) and tissue type (TGase 2) using fluorescence-labeled highly reactive substrate peptides

Abstract

Transglutaminase is a calcium-dependent enzyme that posttranslationally modifies proteins by cross-linking between glutamine and lysine residues or attachment of a primary amine to specific polypeptide-bound glutamine residues. Eight isozymes play essential roles in various mammalian biological processes. The authors have recently identified 12–amino acid preferred substrate peptide sequences that are highly reactive and act in an isozyme-specific manner. In this study, a rapid, isozyme-specific, and sensitive detection of active keratinocyte type (TGase 1) and tissue type (TGase 2) was successful using fluorescence-labeled peptides. This procedure involved using whole-body sections of a mouse to extensively analyze the tissue distribution of both enzymes that revealed clearly distinct patterns. Strong active TGase 1 was observed in epithelial tissues such as tongue, developing teeth, forestomach, and skin epidermis. Significantly active TGase 2 was observed in various types of tissues as predicted and at particularly higher levels in the intestinal mucosa, muscle membrane, and whole veins in the liver. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Figure 1.

In situ detection of active TGase 1 and TGase 2 in mouse tissue sections. (A) Skin sections from the wild-type (+/+) and TGase 1−null (−/−) mice were reacted with 1 µM FITC-pepK5 in the presence of 5 mM CaCl₂. The differential interference images (DIC), the fluorescence images, and their merged images were obtained. As negative controls, reaction samples using 1 µM FITC-pepK5QN or with addition of 5 mM EDTA were carried out. (B) Liver sections from the wild-type (+/+) and the TGase 2−null (−/−) mice were subjected to in situ reaction using 1 µM FITC-pepT26 or 1 µM FITC-pepT26QN as described in (A). The bars indicate 50 µm.

Figure 2.

In situ reaction patterns of whole-mouse body sections using FITC-pepK5 and FITC-pepT26. Whole-body sections were prepared and stained with hematoxylin and eosin (HE). The sections were reacted with 1 µM FITC-labeled peptides (pepK5, pepK5QN, pepT26, and pepT26QN) in the presence of CaCl₂. (A) Midline and (B) sideline. The bars indicate 1 cm. Nonspecific signals in the pictures for the QN mutants may be due to excrement in the bowel.

Figure 3.

Color images of fluorescence signals detected in the sections reacted with FITC-pepK5 and FITC-pepT26. Based on the fluorescence images of Figure 2, pseudo-color images were produced using ImageJ (version 1.43u). To indicate the fluorescence intensity, we divided level of brightness into six classes (from background to maximum area). The six colors, as shown in the box (left in each picture), indicate the intensity in five grades (white, yellow, orange, purple, dark blue) and black as background. Tissues are indicated as follows: Br, brain; Es, esophagus; Fst, forestomach; H, heart; It, intestine; Ki, kidney; Li, liver; Lu, lung; Sp, spleen; Spi, spine; St, stomach; Te, testis; Th, thymus; Tn, tongue. HE, hematoxylin and eosin. The bars indicate 1 cm.

Figure 4.

Tissue distribution of in situ active area of TGase 1 and TGase 2. Enlarged pictures of hematoxylin and eosin (HE) staining (left), fluorescence imaging of FITC-pepK5 (center), and FITC-pepT26 (right) are shown for each tissue. (A) Tongue: FP, filiform papilla; ML, muscle layer; L, lip, TB, taste bud. Teeth: DP, dental papilla; O, odontoblast; D, dentin; E, enamel; A, ameloblast; Sr, sterate reticulum. Stomach: Fst, forestomach (stratified); GlS, Glandular stomach (monolayer); CL, cornified layer; GP, gastric pits; ML, muscle layer. Intestine: McL, mucosal layer; MsL, muscle layer; IV, intestinal villi; IG, intestinal glands; SM, seronous membrane. Skin: SC, skin cornified layer; E, epidermis; D, dermis; ST, subcutaneous tissue; HF, hair follicle. (B) Liver: PV, interlobular portal vein; Hp, hepatocyte. Spine (bone): M, bone marrow; ID, intervertebral disk; EP, epiphyseal plate. Diaphragm (D): Li, liver; Lu, lung. Testis: St, stroma; SeT, semiferous cells. The bars indicate 200 µm.