We describe a class ofcationic structural proteins that associate specifically with intermediate filaments (IF) but not with other types ofcytoskeletal proteins. These proteins, for which the term filaggrin is introduced, are isolated from the stratum corneum of mammalian epidermis. They are species-distinct proteins; for example, rat and mouse filaggrin have different molecular weights and amino acid compositions, but are nevertheless chemically and functionally very similar. They interact in vitro with the IF of several different types of cells to form large fibers or macrofibrils in which many IF are highly aligned in parallel arrays. Stoichiometric analyses suggest that two molecules offilaggrin bind to each three-chain building block of the IF, possibly by ionic interactions with the coiled-coil et-helical regions of the IF.Intermediate filaments (IF) are ubiquitous constituents of the cytoskeleton of eukaryote cells. However, detailed studies of the IF isolated from a wide variety ofdifferent types ofcells have indicated broad differences in their solubility and immunological properties and in the size and complexity of their constituent subunits (1-3). Nevertheless, the IF studied to date appear to be structurally analogous. They are all a-type fibrous proteins and are composed of a common three-chain building block (4-7).Mammalian epidermis is unusual in that its principal differentiation products are filaments of the IF type (keratin filaments). These are synthesized and deposited intracellularly in the inner living cell layers as bundles or fibrils and eventually form the bulk ofthe terminally differentiated cells ofthe stratum corneum (8). The epidermis also produces significant amounts of another structural protein, which has distinctly different physicochemical properties from the IF. This protein is synthesized in the granular layer as a highly phosphorylated precursor and accumulates in amorphous keratohyalin granules, but it is dephosphorylated to become highly cationic when the granular cells differentiate to form the stratum corneum cells (9). The rat cationic protein, called stratum corneum basic protein (10), histidine-rich protein II (11), or histidine-rich basic protein (12), is thought to function in the stratum corneum as an interfilamentous matrix and thereby contribute to the formation ofthe characteristically insoluble protein complex ofthe epidermis, keratin. Support for this notion has been adduced from experiments in vitro. Mixing ofepidermal keratin IF with the rat cationic protein results in the formation of insoluble fibrous structures, or macrofibrils (12)(13)(14)(15). These macrofibrils consist of large numbers of keratin IF aligned in parallel arrays reminiscent of the keratin fibrils in the stratum corneum.In this paper, we report the isolation and characterization of a functionally similar although chemically distinct protein from mouse epidermis. Both the rat and mouse cationic proteins form macrofibrils when mixed with epidermal and inner root sheath keratin IF a...

In this study we investigated why bloodstream forms of Trypanosoma brucei gambiense cross human brain microvascular endothelial cells (BMECs), a human blood-brain barrier (BBB) model system, at much greater efficiency than do T. b. brucei. After noting that T. b. gambiense displayed higher levels of cathepsin L-like cysteine proteases, we investigated whether these enzymes contribute to parasite crossing. First, we found that T. b. gambiense crossing of human BMECs was abrogated by N-methylpiperazine-urea-Phe-homopheylalanine-vinylsulfone-benzene (K11777), an irreversible inhibitor of cathepsin L-like cysteine proteases. Affinity labeling and immunochemical studies characterized brucipain as the K11777-sensitive cysteine protease expressed at higher levels by T. b. gambiense. K11777-treated T. b. gambiense failed to elicit calcium fluxes in BMECs, suggesting that generation of activation signals for the BBB is critically dependant on brucipain activity. Strikingly, crossing of T. b. brucei across the BBB was enhanced upon incubation with brucipain-rich supernatants derived from T. b. gambiense. The effects of the conditioned medium, which correlated with ability to evoke calcium fluxes, were canceled by K11777, but not by the cathepsin B inhibitor CA074. Collectively, these in vitro studies implicate brucipain as a critical driver of T. b. gambiense transendothelial migration of the human BBB.

Trypanosoma brucei contains a soluble serine oligopeptidase (OP-Tb) that is released into the host bloodstream during infection, where it has been postulated to participate in the pathogenesis of African trypanosomiasis. Here, we report the identification of a single copy gene encoding the T. brucei oligopeptidase and a homologue from the related trypanosomatid pathogen Leishmania major. The enzymes encoded by these genes belong to an emerging subgroup of the prolyl oligopeptidase family of serine hydrolases, referred to as oligopeptidase B. The trypanosomatid oligopeptidases share 70% amino acid sequence identity with oligopeptidase B from the intracellular pathogen Trypanosoma cruzi, which has a demonstrated role in mammalian host cell signaling and invasion. OP-Tb exhibited no activity toward the prolyl oligopeptidase substrate H-Gly-Pro-7-amido-4-methylcoumarin. Instead, it had activity toward substrates of trypsin-like enzymes, particularly those that have basic amino acids in both P 1 and P 2 (e.g. benzyloxycarbonyl-Arg-Arg-7-amido-4-methylcoumarin k cat /K m ‫؍‬ 529 s ؊1 M ؊1). The activity of OP-Tb was enhanced by reducing agents and by polyamines, suggesting that these agents may act as in vivo regulators of OP-Tb activity. This study provides the basis of the characterization of a novel subgroup of serine oligopeptidases from kinetoplastid protozoa with potential roles in pathogenesis.

The neurological manifestations of sleeping sickness in man are attributed to the penetration of the blood-brain barrier (BBB) and invasion of the central nervous system by Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. However, how African trypanosomes cross the BBB remains an unresolved issue. We have examined the traversal of African trypanosomes across the human BBB using an in vitro BBB model system constructed of human brain microvascular endothelial cells (BMECs) grown on Costar Transwell inserts. Human-infective T. b. gambiense strain IL 1852 was found to cross human BMECs far more readily than the animal-infective Trypanosoma brucei brucei strains 427 and TREU 927. Tsetse fly-infective procyclic trypomastigotes did not cross the human BMECs either alone or when coincubated with bloodstreamform T. b. gambiense. After overnight incubation, the integrity of the human BMEC monolayer measured by transendothelial electrical resistance was maintained on the inserts relative to the controls when the endothelial cells were incubated with T. b. brucei. However, decreases in electrical resistance were observed when the BMEC-coated inserts were incubated with T. b. gambiense. Light and electron microscopy studies revealed that T. b. gambiense initially bind at or near intercellular junctions before crossing the BBB paracellularly. This is the first demonstration of paracellular traversal of African trypanosomes across the BBB. Further studies are required to determine the mechanism of BBB traversal by these parasites at the cellular and molecular level.

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