Hookworms are blood-feeding intestinal parasites of mammalian hosts and are one of the major human ailments affecting ϳ600 million people worldwide. These parasites form an intimate association with the host and are able to avoid vigorous immune responses in many ways including skewing of the response phenotype to promote parasite survival and longevity. The primary interface between the parasite and the host is the excretory/secretory component, a complex mixture of proteins, carbohydrates, and lipids secreted from the surface or oral openings of the parasite. The composition of this complex mixture is for the most part unknown but is likely to contain proteins important for the parasitic lifestyle and hence suitable as drug or vaccine targets. Using a strategy combining the traditional technology of one-dimensional SDS-PAGE and the newer fractionation technology of OFFGEL electrophoresis we identified 105 proteins from the excretory/secretory products of the blood-feeding stage of the dog hookworm, Ancylostoma caninum. Highly represented among the identified proteins were lectins, including three C-type lectins and three ␤-galactoside-specific S-type galectins, as well as a number of proteases belonging to the three major classes found in nematodes, aspartic, cysteine, and metalloproteases. Interestingly 28% of the identified proteins were homologous to activation-associated secreted proteins, a family of cysteine-rich secreted proteins belonging to the sterol carrier protein/Tpx-1/Ag5/ PR-1/Sc-7 (TAPS) superfamily. Thirty-four of these proteins were identified suggesting an important role in host-parasite interactions. Other protein families identified included hyaluronidases, lysozyme-like proteins, and transthyretin-like proteins. This work identified a suite of proteins important for the parasitic lifestyle and provides new insight into the biology of hookworm infection.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane. The ␣-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the ␤-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give ␣-␤ monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the ␣-␣ dimer bond Cys 524 . In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the ␤-chain residues Cys 798 and Cys 807 and between the ␣-chain Cys 647 and the ␤-chain Cys 872 . The latter bond is the sole ␣-␤ link in the molecule and implies a side-byside alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional ␣-␣ dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys 884 exists as a buried thiol in the soluble ectodomain. The insulin receptor (IR)1 is a homodimer, (␣␤) 2 , held together by disulfide bonds. Each ␣␤ monomer comprises an ␣-chain that is entirely extracellular and a ␤-chain that spans the cell membrane via a single transmembrane link to an intracellular segment that includes the protein-tyrosine kinase domain and the domains involved in binding signal transduction proteins (1). The ␣␤ monomer of the IR is coded for by 22 exons (2), is synthesized with a 27-residue signal sequence, and is glycosylated, S-S linked, and proteolytically processed in that order during transport to the cell surface (3, 4). The extracellular region of the mature receptor (the ectodomain) contains the ␣-chain (1-735) and 194 residues of the ␤-chain (the ␤Ј-chain, residues 736 -929). Two isoforms of the IR, which arise by alternative splicing of the mRNA, are expressed in a tissue-specific fashion; they differ by the presence (plus exon 11) or the absence (minus exon 11) of a 12-residue segment close to the C terminus of the ␣-chain (5).The ectodomain of the IR has been shown (6, 7) to contain two homologous domains (L1 and L2) separated by a single cysteine-rich region (residues 159 -310). The L2 domain is joined by a connecting domain (residues 471-593) to the Cterminal portion (from residue 594) that is comprised of two fibronectin (Fn) III repeats, the first of which contains an insert domain that includes both the ␣-␤ cleavage site and the alternately spliced exon 11 (8, 9). A schematic diagram showing the relative arrangement of the domains is shown in Fig. 7.The ␣-chain has a total of 37 cysteine residues, whereas the ␤-chain has 4 extracellular and 6 intracellular cysteine residues. Homologous re...

The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of Pf-GAPDH with a K i value of 0.2 M, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (K i > 25 M) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a K i value of 1 M, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.