Expression and Function of a Family of Transmembrane Kinases from the Protozoan Parasite Entamoeba histolytica

Abstract

The signaling proteome of Entamoeba histolytica is made of transmembrane kinases (TMKs) that are rarely found in unicellular eukaryotes. There are 90 TMK genes reported for E. histolytica, and these have been grouped into nine distinct families based on motifs present on both extracellular and kinase domains. Of these, the B1 family was chosen for further analysis. Genomic sequencing revealed the presence of 28 members belonging to this family. Genes corresponding to the majority of these were truncated and not considered for further analysis. Only five members were full length and contained both extracellular and cytosolic kinase domains. BLAST analysis revealed the presence of homologs of these B1 TMKs in the nonpathogenic Entamoeba dispar. However, the ligand binding domains of the orthologous B1 TMKs of the two species showed considerable divergence, indicating the possibility of a correlation with the pathogenic potential of the organism. Only two of the five full-length copies (B1.I.1 and B1.I.2) were expressed in E. histolytica under the culture conditions used. Antisera generated against the extracellular domain of B1.I.1 stained the cell surface, particularly the areas of contact between the trophozoites. Staining was also seen in the frontal and posterior regions of the motile amoeba. An amoebic cell line expressing a truncated version of the B1.I.1 that lacked the kinase domain was generated. Inducible expression of the truncated TMK resulted in a decrease in cellular proliferation and an increase in sensitivity to serum starvation. Our data indicate that the B1

Members of the Entamoeba histolytica transmembrane kinase family play non-redundant roles in growth and phagocytosis

“…Expression of PaTMK and sub-family B 1 TMKs has been examined at the protein level (Mehra et al, 2006; Boettner et al, 2008) however studies have been limited to Western blotting and microscopy. Consequently, it is unknown whether TMKs are heterogeneously expressed by trophozoites within a population.…”

“…When the TMKs were divided into nine sub-groups (A, B 1–3 , C, D 1–2 , E and F) based on signature motifs found within the substrate recognition regions of their kinase domains, similarity in the extracellular domains became apparent within sub-groups with respect to the size and the distribution of cysteine-rich motifs, suggesting that TMK subgroups represent functionally distinct receptor families with sub-family-specific substrates and ligands (Beck et al, 2005). However, only two TMKs have been partially characterized to date: PaTMK (TMK96, Subfamily B 3 ) is expressed at the cell surface and functions in erythrophagocytosis (Boettner et al, 2008) and members of the B 1 family of TMKs play a role in proliferation and sensitivity to serum-derived growth factors (Mehra et al, 2006). …”

Molecular Basis of Pathogenesis in Amoebiasis

“…These have been categorized into nine families and a few have been functionally characterized. Members of EhTMKB1 family, such as EhTMKB1-9 and EhTMKB1-2, have been shown to be involved in cellular proliferation and serum response [74,75]. PATMK (EhTMKB3-96) co-localized with human erythroc yt e s at th e s i t e o f co nt ac t a n d i s i nv ol ve d i n erythrophagocytosis and was identified during screening for proteins involved in the recognition and ingestion of apoptotic cells [76•].…”

Protein Kinases of the Parasitic ProtistEntamoeba histolytica

“…These studies have shown the role of this family in serum responsiveness and proliferation. While TMKB1-3 is constitutively expressed at a low level, the expression of TMKB1-9 appears to be regulated by serum, being low in serumstarved cells and induced to high levels after the addition of serum [73,74]. In fact, TMKB1-9 is the major TMKB1 family member to be expressed (ca.…”

“…The automatic curation was performed by Gblock and tree building by PhyML. The significant bootstrap values above 50% are shown[74].…”