Trefoil Factor Family (TFF) Modules Are Characteristic Constituents of Separate Mucin Complexes in the Xenopus laevis Integumentary Mucus: In Vitro Binding Studies with FIM-A.1.

René Stürmer,Jana Reising,Werner Hoffmann

doi:10.3390/ijms21072400

Abstract

The skin of the frog Xenopus laeevis is protected from microbial infections by a mucus barrier that contains frog integumentary mucins (FIM)-A.1, FIM-B.1, and FIM-C.1. These gel-forming mucins are synthesized in mucous glands consisting of ordinary mucous cells and one or more cone cells at the gland base. FIM-A.1 and FIM-C.1 are unique because their cysteine-rich domains belong to the trefoil factor family (TFF). Furthermore, FIM-A.1 is unusually short (about 400 amino acid residues). In contrast, FIM-B.1 contains cysteine-rich von Willebrand D (vWD) domains. Here, we separate skin extracts by the use of size exclusion chromatography and analyze the distribution of FIM-A.1 and FIM-C.1. Two mucin complexes were detected, i.e., a high-molecular-mass Complex I, which contains FIM-C.1 and little FIM-A.1, whereas Complex II is of lower molecular mass and contains the bulk of FIM-A.1. We purified FIM-A.1 by a combination of size-exclusion chromatography (SEC) and anion-exchange chromatography and performed first in vitro binding studies with radioactively labeled FIM-A.1. Binding of 125I-labeled FIM-A.1 to the high-molecular-mass Complex I was observed. We hypothesize that the presence of FIM-A.1 in Complex I is likely due to lectin interactions, e.g., with FIM-C.1, creating a complex mucus network.

Highlights

For a long time, Xenopus laevis skin secretions have been extensively studied because they are a rich source for biologically active peptides, which are known for their hormone-likeand antimicrobial activities
frog integumentary mucin (FIM)-A.1 mainly appeared in Complex II, and only a minority was present in Complex I (Figure 2B)
FIM-A.1 and FIM-C.1 are of different sizes (Figure 2C,F), and size-exclusion chromatography (SEC) using the S-500 column is capable of separating these mucins into Complexes I and II (Figure 2B)

Summary

Introduction

Antimicrobial activities (such as PGLa/PYLa [2]) These peptides are synthesized in granular/serous glands [3], which are more frequent in the dorsal part of the skin. X. laevis protects its skin from infections by antimicrobial peptides and with a mucus barrier, which prevents attachment of microbia and supports the clearance of microorganisms. Knockdown of the skin mucin MucXS (formerly: Otogelin-like) in X. tropicalis tadpoles led to increased susceptibility to infection of these animals with Aeromonas hydrophilia [6]. The frog integumentary mucus is expected to be a complex mixture of a huge variety of proteins, including gel-forming mucins; the latter are typical secretory products of the skin mucous glands [7].

Methods

Results

Conclusion