The surface trans-sialidase family of Trypanosoma cruzi.

KEY WORDS: lectin, glycosyltransferase, immunity, invasion, Chagas' disease, sialyltransferase

CONTENTS

 
INTRODUCTION                                                   386 
SIALIDASE ACTIVITIES IN TRYPANOSOMA CRUZI                      388 
ENZYMATIC PROPERTIES OF THE TRANS-SIALIDASE                    389 
  Sialidase and Trans-Sialidase                                389 
  Donor and Acceptor Specificity                               390 
  Sialidase Activity in Trypanosoma rangeli                    392 
  The Trans-Sialidase of Trypanosoma brucei                    392 
STRUCTURE OF THE TRANS-SIALIDASE FAMILY                        392 
  Motifs that Characterize the Trans-Sialidase Family          393 
  Role of the Carboxy-Terminal Repetitive Motif in 
  Trans-Sialidase                                              396 
  Trans-Sialidase-Family Members Are Probably GPI Anchored     397 
  Evolution of the Trans-Sialidase Family                      398 
  Regulation of Trans-Sialidase-Family Gene Expression         398 
INTERACTIONS OF TRYPANOSOMA CRUZI WITH THE HOST                400 
  Biological Roles of Sialic Acids                             400 
  Modulation of Cell-Surface Sialic Acid and Trans-Sialidase 
  Activity During the 
               Life Cycle of Trypanosoma cruzi                 401 
  A Serum Sialidase Inhibitor (Cruzin)                         403 
  Trans-Sialidase-Related Surface Glycoproteins of 
  Trypanosoma cruzi                                            403 
PRESENT UNCERTAINTIES AND FUTURE DIRECTIONS                    405 

Trypanosomes cannot synthesize sialic acids. Infectious stages of the life cycle of the human pathogen Trypanosoma cruzi express a cell-surface glycolipidanchored trans-sialidase, which can transfer sialic acid between glycoconjugates. Sialic acid is transferred from host cell-surface and serum sialylglycoproteins to trypanosome cell-surface glycoconjugates. The transfer reaction is specific for donors with terminal [alpha]-2,3-linked sialic acid, and terminal [beta]-1,4-linked galactose is the preferred acceptor. In the absence of an acceptor, the enzyme acts as a hydrolase, but cleavage is less efficient than transfer. Trans-sialidase activity is attributable to a few members of a large family of T. cruzi surface glycoproteins, many of which are simultaneously expressed. The functions of the trans-sialidase surface glycoprotein family are unknown but may be important for adhesion, invasion, virulence, or pathogenicity. A trans-sialidase is also expressed in the procyclic forms of Trypanosoma brucei.

INTRODUCTION

Trypanosoma cruzi is the protozoan hemoflagellate responsible for Chagas' disease, which is largely restricted to the indigent population of Central and South America, where it remains an ever-present threat of serious disease. The parasite, unlike its African relative Trypanosoma brucei, does not undergo antigenic variation via a variant surface glycoprotein (VSG) multigene family (20). T. cruzi escapes from the humoral immune response by invading and multiplying within host cells. Infection is characterized by an acute phase, with high parasitemia, followed by an asymptomatic phase, which may last for more than 10 years, leading to chronic disease, with characteristic megasyndromes of the heart and digestive tract (reviewed in 8, 44).

T. cruzi has a complex life cycle (Figure 1), with several biochemically and morphologically distinct stages in the insect and mammalian hosts. Infection of the mammalian host occurs through feces contamination of a wound or mucous membrane by blood-sucking reduviid bugs, such as Rhodnius and Triatoma species, which deposit the infective flagellated metacyclic trypomastigote form. Metacyclic trypomastigotes cannot divide and must enter host tissue cells to differentiate and multiply. T. cruzi is a promiscuous parasite--it can invade a wide rang of cells in vitro. After invasion, the trypomastigotes escape from the parasitophorous vacuole and differentiate into nonflagellated amastigotes, which multiply extensively in the host-cell cytoplasm. Subsequently, they redifferentiate into flagellated trypomastigotes, which do not divide but are released and disseminate the infection until the immune system is able to restrict it. After the insect vector has ingested blood infected with trypomastigotes, these forms differentiate into dividing epimastigotes, which are not infective to mammals. In the later stages of the midgut infection, epimastigotes differentiate once more into infective metacyclic trypomastigotes. This transformation can be mimicked in axenic culture (13). Blood transfusions also provide a significant route of infection in endemic and nonendemic regions (97).

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Chagas' disease sensu stricto is associated exclusively with degeneration of tissues that are innervated by the autonomic nervous system, including a reduction in the number of ganglion cells, and the disease may have a strong autoimmune component. The source and significance of autoimmune responses in Chagas' disease are the subject of substantial research and debate (22)(67)(102).

The surface of T. cruzi has undergone intense scrutiny for glycoproteins and glycolipids that may be involved in parasite differentiation, nutrition, host cell attachment, and penetration. Recent work has identified a sialidase/transsialidase activity, which may modulate the adhesion and penetration of host cells by controlled sialylation of parasite surface glycoconjugates and/or modulation of sialic acid linkages on host cell glycoconjugates. Sialidase activity may also be involved in the exit of intracellular parasites from parasitophorous vacuoles into the cytoplasm, where they are free to divide (34). The purpose of this review is to describe background work on the T. cruzi trans-sialidase and its biochemical properties and structure, and to discuss possible functions of the large multigene family that encodes an array of trans-sialidase-related surface glycoproteins, with molecular weights ranging from approximately 80 to > 220.

SIALIDASE ACTIVITIES IN TRYPANOSOMA CRUZI

Sialidase activity was first recognized in T. cruzi in a sensitive assay in which Pereira (61) detected galactose residues, exposed after incubation of erythrocytes with live trypanosomes or lysates, by their reaction with peanut agglutinin. Subsequent studies showed that the activity was developmentally regulated and probably attributable to a polymorphic family of molecules, ranging in [M.sub.r] from 160,000 to > 220,000 (73)(77). Sialidase activity was absent from amastigotes, but was present in the infective-bloodstream and tissue culture-derived trypomastigotes (Figure 1). The activity in cultures of the noninfective epimastigote stage was 7- to 15-fold lower than that of tissue-culture trypomastigotes (35)(61). Recent studies suggest that epimastigotes contain an enzyme with distinct molecular characteristics (see below). There is disagreement (35)(61) about the level of sialidase activity in epimastigote-derived metacyclic trypomastigotes, which may depend on parasite strain or culture conditions.

Higher levels of sialidase activity in the infectious stages of the life cycle implicated the enzyme in processes specific to the infective forms, such as host-cell penetration or tissue pathogenesis. Although a causal relationship has not been demonstrated, certain myotropic strains of T. cruzi have increased sialidase activity (63). The ability of solubilized T. cruzi sialidase to release free sialic acid from rat myocardial and human vascular epithelial cells also suggested a possible role in host-parasite interactions (63).

Harth et al (35) purified a 60-kDa sialidase activity to apparent homogeneity. The source of their material, metacyclic trypomastigotes derived from an aged epimastigote culture subjected to separation by metrizamide gradients and anion-exchange chromatography, was different from the tissue-culture, cell-derived trypomastigotes used by Cavallesco & Pereira (11), and this variation may have contributed to differences in the findings of the two groups. Cavallesco & Pereira (11) did not purify the T. cruzi sialidase but cut a band showing the sialidase activity from a nondenaturing gel of enzyme released from tissue culture--derived trypomastigotes, and used this to immunize a rabbit. The resulting polyclonal antiserum showed antisialidase activity and identified a set of proteins ranging from 79 to 200 kDa. The sialidase itself could not be positively identified because the antiserum was insufficiently specific, but activity was restricted to the larger components. Monoclonal antibodies that precipitated sialidase activity from cell extracts or culture supernatants identified multiple bands in the range 121--220 kDa in tissue culture--derived trypomastigotes but recognized nothing in epimastigotes or metacyclic trypomastigotes (73).

This work suggested that antibodies to sialidase either cross-reacted with numerous proteins without sialidase activity or that several high-molecular-weight components exhibited sialidase activity. We shall see from subsequent work that the former is probably the case. At no time did Pereira report the identification of a band of 60 kDa and, in view of the purification protocol described by Harth et al (35), the 60-kDa component may have been a degradation product, containing the enzymatically active domain from a larger protein. Nevertheless, there is other evidence of a smaller epimastigote sialidase, which is not phosphatidylinositol-specific phospholipase-C (PIPLC)-susceptible and does not react with the trypomastigote sialidase monoclonal antibodies (86); this may have been the protein purified by Harth. Ultrastructural studies (72)(99) suggested that both groups were working with different proteins because antibodies to each gave quite different labeling patterns in immunoelectron microscopy. These differences might be due to antibodies to contaminants in the purified sialidase or to the extensive sialidase-related gene family now recognized in T. cruzi (see below).

Recent work (9) shows that epimastigotes express a sialidase that has specificity and kinetic properties that are indistinguishable from the trypomastigote enzyme, but this sialidase is structurally distinct in important ways. It lacks the immunodominant carboxy-terminal repetitive domain characteristic of the trypomastigote enzyme (see below), and it is smaller (90 kDa) and monomeric. Also, we can conclude that the epimastigote enzyme is a distinct molecular species that does not result from contamination of the cultures with metacyclic forms because its activity plateaus in early stationary phase when the numbers of metacyclic forms are increasing.

ENZYMATIC PROPERTIES OF THE TRANS-SIALIDASE

Sialidase and Trans-Sialidase

Independent studies identified sialic acid in T. cruzi epimastigotes, even though the parasite appeared incapable of sialic acid synthesis (12)(91). It was suggested some time ago that, because sialylation depended on the presence of serum glycoconjugates and free sialic acids could not be incorporated into the parasite, the sialic acid found on the surface of T. cruzi could result from a cell-surface trans-sialidase activity (71)(122). Sialyltransferases are normally located in the Golgi and use cytidine monophosphate (CMP)-sialic acid as donor, but evidence has been presented for the existence of cell-surface glycosyltransferases, including sialyltransferase, in mammalian cells (89)(97a)(117). That the T. cruzi sialidase and transsialidase activities were probably attributable to the same molecule was implied by coprecipitation of these activities with antibodies specific for a repetitive peptide motif found in some members of the trans-sialidase family (see below) (57)(95). These antibodies can precipitate 50--85% of the total solublizable sialidase activity of T. cruzi. Studies with recombinant enzymes, expressed in Escherichia coli, prove that sialidase and trans-sialidase activities can be attributed to a single molecule (106).

Donor and Acceptor Specificity

Several natural sialoglycoconjugates are susceptible to the sialidase (57)(61)(95), and 4-methylumbelliferyl N-acetyl neuraminic acid is …