Degradations of human immunoglobulins and hemoglobin by a 60 kDa cysteine proteinase of Trichomonas vaginalis

Abstract

The present study was undertaken to investigate the role of cysteine proteinase of Trichomonas vaginalis in escaping from host defense mechanism. A cysteine proteinase of T. vaginalis was purified by affinity chromatography and gel filtration. Optimum pH for the purified proteinase activity was 6.0. The proteinase was inhibited by cysteine and serine proteinase inhibitors such as E-64, NEM, IAA, leupeptin, TPCK and TLCK, and also by Hg2+, but not affected by serine-, metallo-, and aspartic proteinase inhibitors such as PMSF, EDTA and pepstatin A. However, it was activated by the cysteine proteinase activator, DTT. The molecular weight of a purified proteinase was 62 kDa on gel filtration and 60 kDa on SDS-PAGE. Interestingly, the purified proteinase was able to degrade serum IgA, secretory IgA, and serum IgG in time- and dose-dependent manners. In addition, the enzyme also degraded hemoglobin in a dose-dependent manner. These results suggest that the acidic cysteine proteinase of T. vaginalis may play a dual role for parasite survival in conferring escape from host humoral defense by degradation of immunoglobulins, and in supplying nutrients to parasites by degradation of hemoglobin.

Fig. 1

SDS-PAGE (12%) analysis of a cysteine proteinase (arrow) purified from crude extract of Trichomonas vaginalis according to sequential chromatographic steps. M, marker; lane 1, active peaks from Bacitracin-Sepharose 4B affinity chromatography; lane 2, active peaks from Sephacryl S-200 HR gel filtration.

Fig. 2

Degradation of human serum IgG by the purified 60 kDa cysteine proteinase. M, markers; C, serum IgG; lane 1-3, serum IgG incubated with 1, 2 and 5 µg enzyme for 5 hr; lane 4 & 5, serum IgG incubated with 2 µg enzyme for 1 and 2 hr. SDS-PAGE (12%) of IgG and immunoblot with goat anti-human Fc IgG were done. The cysteine proteinase digested heavy chain of serum IgG in a dose-dependent manner (lane 1-3) and a time-dependent manner (lane 4 & 5). Heavy chain (→) is degraded into 35 kDa (★), 32 kDa (▶) and 27 kDa (□).

Fig. 3

Degradation of human IgA (A) and secretory IgA (B) by the 60 kDa cysteine proteinase. M, markers; C, serum IgA (A) or secretory IgA (B); lane 1-3, serum IgA (A) or secretory IgA (B) incubated with 1, 2 and 5 µg enzyme for 5 hr; lane 4-8, serum IgA (A) or secretory IgA (B) incubated with 2 µg enzyme for 1, 2, 5, 12 and 24 hr. SDS-PAGE (12%) of IgA or secretory IgA and immunoblot with sheep anti-human Fc IgA were done. The cysteine proteinase digested heavy chain of serum IgA in a dose-dependent manner (lane 1-3) and a time-dependent manner (lane 4-8). Heavy chain of serum IgA (A) (→) is degraded into 48 kDa (■), 45 kDa (★) and 35 kDa (▶), and that of secretory IgA (B) (→) is degraded into 48 kDa (★).

Fig. 4

Degradation of hemoglobin by the 60 kDa cysteine proteinase (➡ dimer, → monomer of hemoglobin). The enzyme digested hemoglobin in a dose-dependent manner (lane 1-4). M, marker; C, control hemoglobin; lane 1-4, hemoglobin incubated with 1, 2, 5, and 10 µg enzyme for 12 hr.