Effects of GPI-anchored TNAP on the dynamic structure of model membranes

Abstract

Tissue-nonspecific alkaline phosphatase (TNAP) plays a crucial role during skeletal mineralization, and TNAP deficiency leads to the soft bone disease hypophosphatasia. TNAP is anchored to the external surface of the plasma membranes by means of a GPI (glycosylphosphatidylinositol) anchor. Membrane-anchored and solubilized TNAP displays different kinetic properties against physiological substrates, indicating that membrane anchoring influences the enzyme function. Here, we used Electron Spin Resonance (ESR) measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPI-anchored TNAP with model membranes. The goal was to systematically analyze the ESR data in terms of line shape changes and of alterations in parameters such as rotational diffusion rates and order parameters obtained from non-linear least-squares simulations of the ESR spectra of probes incorporated into DPPC liposomes and proteoliposomes. Overall, the presence of TNAP increased the dynamics and decreased the ordering in the three distinct regions probed by the spin labeled lipids DOPTC (headgroup), and 5- and 16-PCSL (acyl chains). The largest change was observed for 16-PCSL, thus suggesting that GPI-anchored TNAP can give rise to long reaching modifications that could influence membrane processes halfway through the bilayer.

Biophysical aspects of biomineralization

“…Garcia et al (2015) used electron spin resonance measurements along with spin labeled phospholipids to probe the possible dynamic changes prompted by the interaction of GPIanchored TNAP with model membranes. In this study, these authors showed that TNAP is probably close to the membrane surface, suggesting that this proximity can be related to the modulation of TNAP activity by the lipid composition of the vesicles, as previously reported (Bolean et al 2010(Bolean et al , 2011Ciancaglini et al 2012;Garcia et al 2015;Simao et al 2010a).…”

The two sides of a lipid-protein story

“…More importantly, lipid membranes, particularly those composed of d i p a l m i t o y l p h o s p h a t i d y l c h o l i n e ( D P P C ) a n d dipalmitoylphosphatidylserine, play a crucial role in the biomineralization process (Simão et al 2010), which highlights the relevance of studies on TNAP-membrane interactions. Garcia et al (2015) investigated the unknown effects of membrane-embedded TNAP on the structural organization of DPPC liposomes as model membranes. The authors were interested in addressing two questions: (1) is the GPI anchor indeed and the only responsible for TNAP association to membranes; and (2) what are the changes on the lipid structural dynamics upon TNAP incorporation?…”

Sistemas miméticos de vesículas da matriz: correlação entre microambiente lipídico e a atividade da fosfatase alcalina no processo de biomineralização

“…A TNAP exibe Atividade fosfomonohidrolítica, que gera fosfato inorgânico (Pi) a partir de pirofosfato inorgânico (PPi) e adenosina-5-trifosfato (ATP) e tem um papel crucial na calcificação. A TNAP está ligada a membranas de células e MVs através de uma âncora de glicosilfosfatidilinositol (Ancorado por GPI), que fornece à enzima mobilidade lateral em relação à membrana (Garcia et al, 2015). Essa mobilidade permite que o TNAP se acumule em microambientes lipídicos específicos associados a microdomínios ricos em esfingolipídios, glicoesfingolipídeos, e colesterol (Chol) (Pizauro et al, 1995;Sakamoto et al, 2013;Mouritsen e Bagatolli, 2015;Bhatia et al, 2016) .…”

“…Em um trabalho recente, Garcia 2015(Garcia et al, 2015, relata a importância de se obter mais informações fisiológicas e estruturais sobre TNAP, quando ancorada através da GPI em um ambiente de bicamada lipídica.…”

“…Empregando-se os sistemas vesiculares de lipossomos, cuja metodologia de obtenção foi padronizada em nosso laboratório 2011;Ciancaglini et al, 2012;Simao et al, 2013;Bolean et al, 2015;Garcia et al, 2015;Bolean et al, 2017a;Bolean et al, 2017b;Favarin et al, 2017), o envolvimento da enzima e dos lipídios durante o processo de mineralização foi mais fácil de ser estudado, pois estes sistemas podem mimetizar a função das vesículas tanto em sistemas in vitro bem como in vivo. Neste sentido, esta Tese teve como principais objetivos a construção de sistemas de lipossomos utilizando em sua composição os lipídios DPPC, DMPC, DOPC e Chol (bem como análogos), em diferentes proporções, e assim reconstituir a TNAP aos sistemas vesiculares formando assim os proteolipossomos.…”