Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii

Abstract

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape

“…In a more recently published report, Paparelli et al [94] have identified a chloroplast-located AP gene NANA whose misexpression or overexpression not only influences photosynthetic carbohydrate metabolism but also plastid and nuclear gene expression [94]. So the localization of these AP genes to the Arabidopsis or grape thylakoid membranes raises the possibility that they may fulfill roles as specific processing enzymes in this organelle, or participate in maintenance or degradation of photosystem proteins [7,95]. …”

Identification and magnetic immobilization of a pyrophilous aspartic protease from Antarctic psychrophilic fungus

“…Aspartic protease (AP, EC 3.4.23), a versatile proteolytic enzyme, can be applied in various industrial processes (Jamdar & Harikumar, 2016 ;Yegin et al, 2011 ). Diverse aspartic proteases from different sources have been reported and characterized (Almeida et al, 2012 ;da Silvaa et al, 2017 ). However, the identification and detailed properties of aspartic proteases from psychrophilic fungi have not been reported till date.…”

Estudio de peptidasas aspárticas de flores de <i>Asteraceae</i> con potencial aplicación biotecnológica

“…En cuanto a su localización subcelular, las PAs de plantas de la familia A1 que han sido caracterizadas, se dirigen principalmente a las vacuolas líticas o al compartimento vacuolar de almacenamiento. Sin embargo, algunas de ellas, se dirigen al retículo endoplasmático o se secretan a la pared celular o al apoplasto (Almeida et al, 2012;González-Rábade et al, 2011;Mazorra-Manzano et al, 2013;van der Hoorn, 2008). Algunos ejemplos de PAs secretadas a la matriz extracelular lo constituyen las PAs de hojas de tabaco 1.…”

“…Los dos motivos de secuencia catalíticos DTG/DSG se encuentran en todas las PAs de plantas pertenecientes a la familia A1 con la excepción de chlapsina, la PA de Chlamydomonas reinhardtii que contiene DTG/DTG al igual que las peptidasas homólogas animales y microbianas (Almeida et al, 2012). En algunas PAs de hongos y de protozoos, los residuos de ácido aspártico catalíticos también se encuentran dentro de los motivos DTG/DSG.…”