Identification of a secreted fatty acid and retinol-binding protein (Hp-FAR-1) from Heligmosomoides polygyrus

Abstract

Hp-FAR-1 is a major, secreted antigen of the parasitic nematode Heligmosomoides polygyrus, a laboratory mouse model frequently used to study the cellular mechanisms of chronic helminth infections. The DNA encoding Hp-FAR-1 was recovered by screening a fourth larval (L₄) H. polygyrus cDNA expression library using antibodies raised against L₄ stage excretory/secretory (E/S) proteins. Predictions of secondary structure based on the Hp-FAR-1 amino acid sequence indicated that an alpha-helix predominates in Hp-FAR-1, possibly with some coiled-coil conformation, with no beta-structure. Fluorescence-based ligand binding analysis confirmed that the recombinant Hp-FAR-1 (rHp-FAR-1) binds the fluorescent fatty acid analog 11-((5-[dimethylaminoaphthalene-1-sulfonyl)amino)undecanoic acid (DAUDA), and by competition oleic acid. RT-PCR amplification of the hp-far-1 gene indicated that the gene is transcribed in all parasitic stages of the organism's life cycle. The presence of a secreted FAR protein in the well-defined laboratory model of H. polygyrus provides an excellent model for the further study and analysis of the in vivo role of secreted FAR proteins in parasitism, and supports the mounting evidence that secreted FAR proteins play a major role in nematode parasitism.

Keywords: Heligmosomoides polygyrus; Hp-FAR-1; host-parasitic relationship; hp-far-1; lifecycle; molecular biology; nematode; retinol binding.

Fig. 1

A consensus casein kinase II phosphorylation site at residues 45-48 (underlined) and secondary structure analysis of Hp-FAR-1 predicts a predominantly alpha-helical conformation. The algorithms applied to the entire alignment predicted 72% helix, 28% loop structures, but no β/extended structure.

Fig. 2

SDS-PAGE and western blot analysis of recombinant Hp-FAR-1 samples. A) Western blot analysis of recombinant Hp-FAR-1 expressed in the pZL1 vector in Escherichia coli DH10B, demonstrating specificity of mouse anti-L₄E/S HIMS for rHp-FAR-1. Lane 1: positive result for pre-purified recombinant Hp-FAR-1 expressed in E. coli DH10B; Lane 2: negative control, pre-purified protein extract from pZL1 vector without Hp-FAR-1 insert in E. coli DH10B; Lane 3: molecular weight marker. B) SDS-PAGE analysis of purified rHp-FAR-1 and GST-Hp-FAR-1. Lane 1: broad range molecular weight markers, Lane 2: purified Hp-FAR-1, Lane 3: purified GST-Hp-FAR-1. C) Western blot analysis probed with anti-GST antibody. Lane 1: broad range molecular weight marker; Lane 2: negative result for rHp-FAR-1 with no GST fusion protein; Lane 3: positive result for GST-Hp-FAR-1 fusion protein at the predicted size of approximately 42 kDa.

Fig. 3

Ligand binding by rHp-FAR-1. Protein was mixed with environment-sensitive fluorescent ligands DAUDA (panel A), oleic acid (panel B) or retinol (panel C). A) Binding of DAUDA by Hp-FAR-1 indicated by a substantial increase in fluorescence emission by DAUDA and the subtraction spectrum showing a significant blue shift in peak emission. B) Highly efficient displacement of DAUDA from rHp-FAR-1 by successive additions of oleic acid. The concentration of compounds in the cuvette were approximately 1 μM DAUDA, and approximately 0.08 μM, 0.9 μM, and 9 μM oleic acid in the successive additions. C) Binding of retinol by rHp-FAR-1 indicated by a substantial increase in fluorescence emission intensity when retinol is added to a solution of Hp-FAR-1 in buffer.

Fig. 4

Transcription of Hp-FAR-1 in L₃, L₄ and adult Heligmosomoides polygyrus using Hp-FAR-1 specific primers. RT-PCR was performed on mRNA isolated from the L₃, L₄, and adult stages of H. polygyrus. Lane 1: DNA ladder; Lane 2: no template control; Lane 3: L₃ H. polygyrus cDNA; Lane 4: L₄ H. polygyrus cDNA; Lane 5: Adult H. polygyrus cDNA.