Structural and lipid-binding characterization of human annexin A13a reveals strong differences with its long A13b isoform

Abstract

Annexin A13 is the founder member of the vertebrate family of annexins, which are comprised of a tetrad of unique conserved domains responsible for calcium-dependent binding to membranes. Its expression is restricted to epithelial intestinal and kidney cells. Alternative splicing in the N-terminal region generates two isoforms, A13a and A13b, differing in a deletion of 41 residues in the former. We have confirmed the expression of both isoforms in human colon adenocarcinoma cells at the mRNA and protein levels. We have cloned, expressed, and purified human annexin A13a for the first time to analyze its structural characteristics. Its secondary structure and thermal stability differs greatly from the A13b isoform. The only tryptophan residue (Trp 186 ) is buried in the protein core in the absence of calcium but is exposed to the solvent after calcium binding even though circular dichroism spectra are quite similar. Non-myristoylated annexin A13a binds in a calcium-dependent manner to acidic phospholipids but not to neutral or raft-like liposomes. Calcium requirements for binding to phosphatidylserine are around 6-fold lower than those required by the A13b isoform. This fact could account for the different subcellular localization of both annexins as binding to basolateral membranes seems to be calcium-dependent and myristoylation-independent.

An alternative N-terminal fold of the intestine-specific annexin A13a induces dimerization and regulates membrane-binding

“…S3). Under the conditions that we tested, membrane recruitment was incomplete, consistent with previously published studies (13,16). The ANX A13a S73C (enhanced dimer) had subtly decreased membrane association relative to WT, which may be anticipated because this mutation only modestly increased the percentage of dimer (Fig.…”

“…The structures of ANX A13 isoforms have been proposed to center around a highly conserved core of helical bundles common to all characterized annexins (13,16). Surprisingly, the crystal structure of ANX A13a reveals only three of the four (Fig.…”

“…Cellular localization studies of ANX A13 homologs from rabbit (11) and fluke (12) confirmed the narrow intestine-specific expression pattern. More recently, RT-PCR of two human colorectal carcinoma cell lines showed that both the ANX A13a and ANX A13b isoforms are expressed in cells (13). The enrichment of ANX A13 in vesicles shedding from the lumenal tips of intestinal microvilli (14) suggests a possible biological role in the creation of these exosomelike vesicles.…”

High-Throughput Identification of Calcium Regulated Proteins Across Diverse Proteomes

“…After cation addition, the free concentrations increased to levels that should saturate cation binding domains and favor cation-protein interactions (Fig. S1A) 34 . Under these conditions, we quantified the relative abundance of proteins following the experimental flow shown in Fig 1A . To identify Calcium Regulated Proteins (CRPs), we established a definition for significant thermal stability changes and hit criteria (Fig.…”

Investigating Open Reading Frames in Known and Novel Transcripts using ORFanage

“…Proteins in this family contain two major domains, one at the C-terminus for the Ca 2+ binding effect, and the other at the N-terminus responsible for the membrane interactions. While the core domain at the C-terminus is highly conserved across the gene family, the N-terminus is variable 47 , allowing for tissue-specific regulation 48, 49 and localization 50 .The two known forms of the gene differ only in the length of the last helical structure, where the incorporation of additional peptides allows for an extension of the first helix.…”