Identification and gene expression analysis of a large family of transmembrane kinases related to the Gal/GalNAc lectin in Entamoeba histolytica

Abstract

We identified in the Entamoeba histolytica genome a family of over 80 putative transmembrane kinases (TMKs). The TMK extracellular domains had significant similarity to the intermediate subunit (Igl) of the parasite Gal/GalNAc lectin. The closest homolog to the E. histolytica TMK kinase domain was a cytoplasmic dual-specificity kinase, SplA, from Dictyostelium discoideum. Sequence analysis of the TMK family demonstrated similarities to both serine/threonine and tyrosine kinases. TMK genes from each of six phylogenetic groups were expressed as mRNA in trophozoites, as assessed by spotted oligoarray and real-time PCR assays, suggesting nonredundant functions of the TMK groups for sensing and responding to extracellular stimuli. Additionally, we observed changes in the expression profile of the TMKs in continuous culture. Antisera produced against the conserved kinase domain identified proteins of the expected molecular masses of the expressed TMKs. Confocal microscopy with anti-TMK kinase antibodies revealed a focal distribution of the TMKs on the cytoplasmic face of the trophozoite plasma membrane. We conclude that E. histolytica expresses members of each subgroup of TMKs. The presence of multiple receptor kinases in the plasma membrane offers for the first time a potential explanation of the ability of the parasite to respond to the changing environment of the host.

FIG. 1.

Phylogenetic and sequence analysis of the kinase domain of E. histolytica transmembrane kinases. CMGC is the cyclin-dependent kinase, mitogen-activated protein kinase, glycogen synthase kinase, and CDK-like kinase family. CAMK is the calmodulin-dependent kinase family. AGC is the family of protein kinases A, G, and C. TMKs were aligned to Hanks's alignment (20) using CLUSTALX (61). The aligned sequences were bootstrapped using Seqboot, Protpars, and Consense (15). Significant bootstrap values are shown in bold, and bootstrap values above 50 are shown. The GenBank numbers for the TMKs are in Tables S1 and S2. SplA is GenBank accession no. U32174 (40).

FIG. 2.

Diagram of Igl and the TMKs. This diagram shows the approximate sizes of the different proteins and the distribution of the CXXCXXGYY motifs in the extracellular domain (indicated by a black circle). The CXXCXXGYY motifs are part of a larger motif, CXXCXXG(Y)(Y/F)(L/V/F/Y/M)-Polar-Polar, which also can begin with CXC instead of CXXC. GPI represents a putative GPI anchor. A black rectangle indicates a transmembrane domain, and a black oval indicates a putative kinase domain. The serine (SSS)- and serine/threonine (SSTT)-rich regions found in groups D1 and D2 are shown. Numbers in brackets indicate numbers of known family members.

FIG. 3.

Expression of TMKs during log-phase culture as determined by oligoarrays. The TMKs were classified into groups: A, B2, B3, C, D1, D2, E, F, G, and “other” (tmk58). Expression of jacob (encoding a cyst protein not expected to be expressed in trophozoites), ebp1, and ebp2 genes is shown for reference. TMK genes expressed at a higher level than jacob (P < 0.05) are indicated by an asterisk and were as follows: group A, tmk61 (391.t00004-AAFB01000774, 279.t00010-AAFB01000993), tmk65 (62.t00013-AAFB01000240), and tmk72 (302.t00003-AAFB01000819); group B2, tmk02 (70.t00014-AAFB01000264), tmk08 (10.t00040-AAFB01000051), and tmk74 (6.t00088-AAFB01000031); group B3, tmk21 (42.t000019-AAFB01000175), and tmk28 (66.t00027-AAFB01000251); group C, tmk39 (359.t00009-AAFB01000933), and tmk63 (20.t00067-AAFB01000094); group D1, tmk40 (65.t00015-AAFB01000247), and tmk56 (5.t00091-AAFB01000028); group D2, tmk19 (135.t00017-AAFB01000458), tmk44 (159.t00012-AAFB01000511), and tmk46 (131.t00015-AAFB01000449); group E, tmk22 (12.t00043-AAFB01000464) and tmk54 (75.t00011-AAFB01000285); group F, tmk59 (304.t00008-AAFB01000821); and group G, tmk06 (274.t00010-AAFB01000764). Error bars represent the standard error of the mean of three hybridizations (biological replicates).

FIG. 4.

Expression of TMK family genes during culture as determined by real-time PCR. Quantitative real-time PCR was performed on (A) the RNA polymerase II gene (rna pol ii) (27.t00035-AAFB01000114), (B) the RNA polymerase II 13 gene (rna pol ii 13) (344.t00001-AAFB01000903), (C) the RNA polymerase II L gene (rna pol ii l) (147.t0005-AAFB01000482), (D) tmk19 (135.t00017-AAFB01000458), (E) tmk21 (42.t00019-AAFB01000175), (F) tmk63 (20.t00067-AAFB01000094), (G) tmk65 (62.t00013-AAFB01000240), (H) tmk71 (268.t00007-AAFB01000754), (I) tmk79 (71.t00002-AAFB01000266), and (J) tmk98 (361.t00001-AAFB01000937). Two sequential growth curves are shown. For growth curve A (triangles and solid line), samples were collected at 12, 48, 96, and 144 h postinoculation. For growth curve B (squares and dashed line), samples were collected at 12, 24, 48, 72, 96, 120, and 144 h postinoculation. Triplicate samples were collected at each time point. Culture B was established by transferring 300,000 amebae from culture A at 144 h. The standard errors of three biological samples, with each sample analyzed in duplicate, are shown. To allow comparison between time points, data for the TMKs were normalized to the average of RNA polymerase II, RNA polymerase II L, and RNA polymerase II 13. The average expression of these three genes was defined as 1,000 units of expression.

FIG. 5.

Recognition of Entamoeba histolytica surface proteins by anti-ΔTMK96 rabbit serum. (A) Soluble trophozoite proteins as well as whole-cell lysates were analyzed by Western blots with anti-ΔTMK96 rabbit serum (1:5,000 dilution). Preimmune serum did not recognize any trophozoite proteins on Western blots (data not shown). (B) Confocal microscopy of permeabilized trophozoites with anti-ΔTMK96 rabbit serum. (C) Confocal microscopy of permeabilized trophozoites with anti-Gal/GalNAc lectin antibodies. No staining was seen with preimmune rabbit serum or in nonpermeabilized trophozoites with the anti-ΔTMK96 serum (data not shown). Magnification, ×400.