Influence of the 524-VAAEIL-529 sequence of annexins A6 in their interfacial behavior and interaction with lipid monolayers

Highlights

• Annexins A6-1 and A6-2 are calcium- and membrane-binding proteins.

• Annexins A6-2 differs from the A6-1 by the absence of 524-VAAEIL-529 sequence.

• Minor differences in their PM-IRRAS spectra were observed.

• Persistence of the 516–529 α-helix in AnxA6-2 despite the lack of the VAAEIL sequence.

Abstract

Annexin A6 (AnxA6), a calcium- and membrane-binding protein, is expressed in mammalian cells in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7. The different intracellular localization of these two isoforms suggests distinct function in membrane dynamics. The aim of this work was to analyze the behavior of AnxA6 isoforms at the air/water interface alone and in the presence of membrane mimicking lipid monolayers. Using Langmuir technique showed that AnxA6-2 was less adsorbed to the neat air–water interface than AnxA6-1 at acidic pH and minor differences in their PM-IRRAS spectra were observed. Both isoforms exhibited similar behavior towards cholesterol monolayer. However, the interactions of AnxA6-2 with cholesterol ester monolayer were most favorable compared to AnxA6-1. Our experimental data are discussed in relation with the different intracellular localization of the two isoforms and with our constructed model of AnxA6-2 with the known crystal structure of AnxA6-1 showing the persistence of the 516–529 α-helix in AnxA6-2 despite the absence of the 524-VAAEIL-529 sequence.

Introduction

Annexins are described as calcium sensors and membrane structure organizers [1]. Among them, there is annexin A6 (AnxA6) which is involved in calcium homeostasis, membrane traffic and membrane organization. Due to alternative mRNA splicing of the 21st exon, in mammalian cells, AnxA6 is expressed in two isoforms: AnxA6-1 and AnxA6-2, the latter lacking the 524-VAAEIL-529 sequence at the start of repeat 7 [2], [3], [4]. Several observations suggest that the functions of the two isoforms differ from each other [5], [6]. AnxA6-1 prevails in normal tissues [7] and cells, such as mouse Balb/3T3 fibroblasts, while AnxA6-2 is more abundant in neoplastic cells [8]. In resting conditions, AnxA6-1 is associated with early endosomes and AnxA6-2 with late endosomes [9] and this distribution is sensitive to pH and calcium concentration [9]. This suggests distinct functions of AnxA6 isoforms in membrane dynamics. Furthermore, AnxA6 isoforms may have distinct influence on calcium fluxes. In A431 cells, which lack endogenous AnxA6, the synthesis of exogenous AnxA6-1 inhibits voltage-dependent Ca²⁺ influx upon stimulation with epidermal growth factor [5]. It was also demonstrated that the overexpression of both isoforms affects secretory properties of PC12 cells. For example, the two AnxA6 isoforms interact with different targets engaged in regulation of calcium homeostasis and dopamine secretion in PC12 cells but AnxA6-2 seems to have a stronger impact on these phenomena than AnxA6-1 [6].

All of the studies mentioned above suggest that both isoforms played different role in the cells and their different localizations may be due to different functions or different interactions with the lipids of the cellular compartments. Thus, the aim of this work was to investigate possible differences in the behavior of annexin A6 isoforms at the air–water interface alone or in the presence of biomimetic membranes such as interfacial lipid monolayers.