Polyglycylation of tubulin in the diplomonad Giardia lamblia, one of the oldest eukaryotes

Abstract

We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia, which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of a-tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine-tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin-specific polyglycylation could be as old as tubulin and axonemal structures.Key words: Flagella; Polyglutamylation; Polyglycylation; Post-translational modification; Tubulin; Tyrosination to search for their evolutionary origin because they are unique to tubulin, a typical eukaryotic protein.Based on ultrastructural characteristics and several molecular phylogenies there is general agreement that the diplomonads were among the first branches which emerged from the eukaryotic tree. Diplomonads, like other Archezoa, are thought to have arisen before the acquisition of mitochondria and to have retained many primitive features of the first nucleated cells [17][18][19][20][21]. Giardia lamblia is a particularly wellcharacterized diplomonad. Its cytoskeleton is dominated by microtubules. Giardia has eight flagellar axonemes and a large disc cytoskeleton which consists of rnicrotubules and the microribbons, which arise from ~-giardin [22,23]. Here we report an analysis of the post-translational modifications of ct-tubulin from Giardia based on the characterization of its carboxyterminal peptide.

Biology ofGiardia lamblia

“…The microtubules of the ventral disk, as well as those of the flagella, are presumably composed of ␣␤-tubulin. Posttranslational modifications of the G. lamblia tubulins include acetylation and polyglycylation (310,345,346). Benzimidazoles are compounds used primarily as antihelminthic agents, but they also have significant in vitro activity against G. lamblia as well as in vivo efficacy for giardiasis (49,68,70,122,233).…”

The tubulin code at a glance

“…Moreover, polyglutamylation is prominent on ciliary and flagellar axonemes (Bré et al, 1994;Fouquet et al, 1994;O'Hagan et al, 2011), where it regulates the beating behavior and integrity of these organelles (reviewed in Konno et al, 2012). In contrast to glutamylation, glycylation has so far been exclusively observed on axonemal MTs (Bré et al, 1996;Redeker et al, 1994;Rüdiger et al, 1995;Weber et al, 1996;Xia et al, 2000) and has been implicated in the mechanical stabilization of the axoneme (Pathak et al, 2011;Rogowski et al, 2009;Wloga et al, 2009). …”

Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules inParamecium

“…The physico-chemical analysis of ciliary tubulin from the protist Paramecium led to the discovery of a new polymodification, polyglycylation, which consisted of an additional lateral chain of up to 34 glycine units on both axonemal tubulin subunits . Since then, studies on polyglycylation, using either mass spectrometry (Rü diger et al, 1995;Mary et al, 1996;Multigner et al, 1996;Weber et al, 1996) or two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49 (Bré et al, 1996), have involved principally axonemes of various cell types. Taken together, the data suggest that axonemal tubulin could be the preferred substrate for polyglycylation.…”