Identification of the functional site of erythrocyte protein 4.1 involved in spectrin-actin associations

Peptides produced by mild chymotryptic digestion of human erythrocyte protein 4.1 mimic the ability of intact 4.1 to promote the binding of spectrin to F-actin. This complex-promoting activity was found to reside in an 8-kDa peptide which was fully functional when dissociated from other protein 4.1-derived peptides, indicating that noncovalent complexes of multiple peptides were not essential for activity. The 8-kDa peptide was incorporated into a ternary complex with spectrin and F-actin in approximately stoichiometric amounts. Amino acid composition and two-dimensional peptide mapping show that the 8-kDa active peptide is located within the 10-kDa region of protein 4.1 which contains a cAMP-dependent phosphorylated site.     

The 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure

The primary structure of the 43-kilodalton peripheral membrane protein (43-kDa protein) of Torpedo nicotinic postsynaptic membrane has been determined. The 43-kDa protein, which was isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has an amino terminus resistant to Edman degradation, while the sequence at the carboxyl terminus is Tyr-Val. An amino acid sequence of 405 residues was obtained by NH2-terminal sequence analysis of complementary peptides generated by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and endoproteinase Lys-C, as well as by chemical cleavage at methionine. This sequence of molecular mass 45,618 daltons lacks the amino terminus but extends to the carboxyl terminus of the 43-kDa protein. Unusual structural features of the 43-kDa protein include two regions of approximately 80 residues, each containing 10% cysteine, as well as stretches predicted to exist as amphipathic alpha-helices. Other than the group blocking the amino terminus, no evidence was found for posttranslational modification of amino acids. The 43-kDa protein may represent a novel protein family because a computer search of this sequence with the National Biomedical Research Foundation data base (Release 12.0) did not reveal any significant homology to known protein sequences.     

The primary structure of the 32-kDa subunit of human replication protein A

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.     

O-N-acetyl-D-glucosamine moiety on discrete peptide of multiple protein 4.1 isoforms regulated by alternative pathways

Erythrocyte protein 4.1 is a cytoplasmic protein that possesses a protein-saccharide modification structure, an O-N-acetyl-D-glucosamine (GlcNAc) moiety. We determined the amino acid sequence of the proteolytic fragment containing the O-GlcNAc moiety after labeling the saccharide with [3H]galactose in the presence of bovine milk galactosyltransferase. Glycosylation appears to occur on one or more serine or threonine residues in the following sequence: Thr-Ala-Gln-Thr-Ile-Thr-Ser-Glu-Thr-Pro-Ser-Ser-Thr-Thr-Thr-Thr-Gln-Ile-Thr-Lys . This sequence corresponds to the carboxyl-terminal half of the 34-amino acid peptide in the 22/24-kDa carboxyl-terminal domain of protein 4.1, which is one of the discrete peptides regulated by alternative RNA splicing. Multiple protein 4.1 isoforms in erythroid and nonerythroid cells including major components of erythrocyte membrane proteins, 4.1a and 4.1b, appear to contain this sequence since most immunochemically reactive proteins were labeled with [3H]galactose, with the exception of several variant polypeptides. These results appear to suggest the functional or biological significance of the O-GlcNAc linkage in protein 4.1.     

Modulation of protein 4.1 binding to inside-out membrane vesicles by phosphorylation

The effect of phosphorylation on the binding of protein 4.1 to erythrocyte inside-out vesicles was investigated. Protein 4.1 was phosphorylated with casein kinase A, protein kinase C, and cAMP-dependent protein kinase. An analysis of the phosphopeptides generated by alpha-chymotryptic and tryptic digestion indicates these kinases phosphorylate similar as well as distinct domains within protein 4.1. All three enzymes catalyze the phosphorylation to varying degrees of the 46-, 16-, and 8-10-kDa fragments derived from limited chymotryptic cleavage. In addition, casein kinase A phosphorylates a 24-kDa domain, whereas protein kinase C phosphorylates a 30-kDa domain. Protein 4.1 phosphorylated by casein kinase A and protein kinase C, but not cAMP-dependent protein kinase, exhibits a reduced binding to KI-extracted inside-out vesicles. On the other hand, phosphorylation of inside-out vesicles by casein kinase A does not affect their ability to bind protein 4.1. The inside-out vesicles, however, inhibit the phosphorylation of protein 4.1 by casein kinase A and protein kinase C, but not by cAMP-dependent protein kinase. These results suggest that casein kinase A and protein kinase C may modulate the binding of protein 4.1 to the membrane by phosphorylation of specific domains of the cytoskeletal protein. Since the 30-kDa domain has been suggested as a membrane-binding site, that phosphorylation by protein kinase C reduces the binding of protein 4.1 to inside-out vesicles is perhaps not surprising. On the other hand, the role of the casein kinase A substrate 24-kDa domain in membrane binding has not been established and needs to be examined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid sequence and molecular characterization of a D-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs

The complete amino acid sequence of a 11.5-kDa subunit of D-galactoside binding lectin purified from sea urchin (Anthocidaris crassispina) eggs is presented. The 105-residue sequence of the subunit was determined by analysis of the intact S-carbamoylmethylated protein and peptides generated by digestion with Achromobacter protease I or Staphylococcus aureus V8 protease. The lectin exists as a disulfide-linked homodimer of two subunits; the dimeric form is essential for hemagglutination activity. However, the monomeric form obtained by partial reduction retains the carbohydrate binding capacity. Neither Ca2+ nor SH reagent is essential for hemagglutination or carbohydrate binding. The sequence has no similarity to that of any known protein and apparently represents a new type of galactoside binding lectin.     

Properties of the C-terminal domain of 4.1 proteins.

At the C-terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C-terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein-protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18-21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr-rich sequence. The CTD was first described as a 22/24-kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem. 259, 4603-4608]. Here we show that in 4.1R the 22/24-kDa fragment is not stable but rapidly processed to a 15-kDa fragment by chymotrypsin. The 15-kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709-858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary-structure predictions indicate that this domain is composed of five or six beta strands with an alpha helix before the most C-terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.     

Domain structure of the basement membrane heparan sulfate proteoglycan

We have used proteolytic digestions and immunological reactivity to map regional domains of the 400-kilodalton (kDa) core protein of the heparan sulfate containing basement membrane proteoglycan from the Englebreth-Holm-Swarm tumor. Digestion with V8 protease caused the rapid release of numerous large peptides ranging in size from 80 to 200 kDa and a 44-kDa peptide. The 44-kDa peptide (P44) was stable to further digestion, but the larger peptides were eventually degraded to a 46-kDa peptide (P46). Both the P44 and P46 fragments migrate slower in the presence of a reducing agent, indicating intrachain disulfide bonding, and do not have heparan sulfate side chains. Antisera to the P46 fragment, however, did not react with P44 fragment, and the amino acid compositions of P46 and P44 fragments were different. This suggests that these two fragments were unrelated. Trypsin digestion of the proteoglycan immediately released a 200-kDa peptide (P200) that also lacked heparan sulfate side chains. Digestion of the P200 fragment with V8 protease produced the P44 and P46 fragments in the same temporal sequence seen with V8 protease digestion of the proteoglycan. Antisera to the P200 fragment reacted strongly with the P44 and P46 fragments. These results show that the P44 and P46 domains are contained within the P200 domain. The rapid release of the P44 domain indicates that it is located at one end of the core protein. The large size of these proteolytic fragments suggests the core protein contains considerable conformational structure, and the absence of heparan sulfate on the P200 domain indicates that the side chains are asymmetrically located on the core.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

A rapamycin-selective 25-kDa immunophilin.

FKBP25, a previously uncharacterized 25-kDa FK506- and rapamycin-binding protein, was purified to homogeneity from calf thymus, brain, and spleen, and the sequence of a 215 amino acid (aa) 24-kDa C-terminal peptide was established. The N-terminal domain (101 aa) is unrelated to any known protein, is hydrophilic, and is predicted by circular dichroism spectroscopy to be largely alpha-helix. The C-terminal domain (114 aa) is homologous to FKBP12 and other FKBPs but has a potential nuclear targeting sequence and a unique insertion of seven amino acids in one of its loops. FKBP25 displays the rotamase activity characteristic of FKBPs; the activity is inhibited by the immunosuppressants rapamycin (Ki = 0.9 nM) and FK506 (Ki = 160 nM), but not cyclosporin A. The protein, its rapamycin selectivity, and the potential nuclear targeting sequence are discussed in terms of the structure of hFKBP12.     

Identification of the DNA-binding domain of the FLP recombinase

We have subjected the FLP protein of the 2-micron plasmid to partial proteolysis by proteinase K and have found that FLP can be digested into two major proteinase K-resistant peptides of 21 and 13 kDa, respectively. The 21-kDa peptide contains a site-specific DNA-binding domain that binds to the FLP recognition target (FRT) site with an affinity similar to that observed for the native FLP protein. This peptide can induce DNA bending upon binding to a DNA fragment containing the FRT site, but the angle of the bend (approximately 24 degrees) is smaller in magnitude than that induced by the native FLP protein (60 degrees). The additional DNA bending induced by the interaction between two native FLP molecules bound to the FRT site is not observed with the 21-kDa DNA-binding peptide. Amino-terminal sequencing has been used to map this peptide to an internal region of FLP that begins at residue Leu-148. It is likely that the DNA-binding peptide includes the catalytic site of the FLP protein.     

Isolation and characterization of a 60-kilodalton glycoprotein esterase from liver microsomal membranes

A glycoprotein having a subunit weight of approximately 60,000 was isolated from rabbit liver microsomes. It is a predominant component of the hepatic microsomal membrane and reacts rapidly with diisopropylphosphorofluoridate (DFP), resulting in the loss of enzymatic activity toward artificial substrates such as acyl esters of o-nitrophenols. Automated Edman degradation of this protein together with sequence analysis of peptides provided the NH2-terminal sequence of some 70 residues as follows: His-Pro-Ser- Ala-Pro-Pro-Val-Val-Asp-Thr-Val-Lys-Gly-Lys-Val- Leu-Gly-Lys-Phe-Val-Ser-Leu-Glu-Gly-Phe-Ala-Gln- Pro-Val-Ala-Val-Phe-Leu-Gly-Val-Pro-Phe-Ala-Lys- Pro-Pro-Leu-Gly-Ser-Leu-Arg-Phe-Ala-Pro-Pro-Gln- Pro-Ala-Glu-Ser-Trp-Ser-His-Val-Lys-Asn (CHO)- Thr-Thr-Ser-Tyr-Pro-Pro-Met-Cys-Ser-Ser. A carbohydrate attachment was identified at asparaginyl residue 61. The COOH-terminal peptide of the protein was isolated from two independent enzymatic digests, and its sequence was established as Arg-Glu-Thr-Glu-His-Ile-Glu-Leu. In order to isolate the DFP binding peptide, liver microsomes were labeled with [3H]DFP and the 60-kDa protein containing covalently bound DFP isolated in pure form. Following reduction and carboxymethylation, the DFP-labeled protein was fragmented with trypsin and the digest subjected to gel filtration. Digestion of the labeled peptide preparations with chymotrypsin followed by chromatography of the digest yielded two diisopropylphosphoryl (DIP) peptides. Automated Edman degradation of these peptides provided the following amino acid sequences: Gly-Glu-DIPSer- Ala-Gly-Gly-Gln-Ser-Val-Ser-Ile-Leu-Leu-Leu-Ser- Pro and Thr-Val-Ile-Gly-Asp-DIPHis-Gly-Asp-Glu-Ile-Phe. The active site serine peptide of the 60-kDa protein shows some 70% similarity to the active center region of choline esterases. While the postulated active histidyl residue in choline esterases has not been identified, it is proposed that the DFP binding histidine of the 60-kDa protein corresponds to His-438/440 of choline esterases.     

The calmodulin-binding site of the plasma membrane Ca2+ pump interacts with the transduction domain of the enzyme.

Calpain proteolysis of the plasma membrane Ca2+ pump removes a C-terminal 14-kDa portion which includes the calmodulin-binding domain. This produces a fully activated 124-kDa fragment, which can be inhibited by synthetic versions of the calmodulin-binding domain. The inhibition is strongest when Trp-8 in the latter domain is replaced by a Tyr residue (Falchetto, R., Vorherr, T., Brunner, J., & Carafoli, E., 1991, J. Biol. Chem. 266, 2930-2936). In the present study, the N-terminus of the 28-residue synthetic calmodulin-binding domain was acetylated with 3H-acetic anhydride, and Phe in position 25 was replaced by a phenylalanine derivatized with a diazirine-based, photoactivatable carbene precursor. This peptide (C28WC*) inhibited the fully active 124-kDa fragment of the pump and became cross-linked to it upon photolysis. After proteolysis of the fragment with Asp-N or Staphylococcus aureus V8 (Glu-C) protease, labeled peptides were isolated by reversed-phase high-performance liquid chromatography and subjected to Edman sequence analysis. The peptides originated from a region of the pump located within the unit protruding into the cytoplasm between transmembrane domain two and three. This unit has been proposed to be the site of the energy transduction domain, which would couple the ATP hydrolysis to Ca2+ translocation.     

Murine T cell-stimulatory peptides from the 19-kDa antigen of Mycobacterium tuberculosis. Epitope-restricted homology with the 28-kDa protein of Mycobacterium leprae.

Fifteen overlapping synthetic peptides, spanning the entire amino acid sequence of the Mycobacterium tuberculosis 19-kDa protein, were used to identify epitopes recognized by murine T cells. Five of the 15 peptides tested were able to elicit in vitro lymph node T cell proliferative responses in C57BL/10 mice primed by footpad inoculation with homologous peptide. Analysis in congenic strains of mice revealed H-2 restriction in the response to four peptides. However, one peptide, 19.7 (residues 61 to 80), induced T cell responses in all four haplotypes tested. This peptide was also unique in being able to stimulate lymph node cells from C57BL/10 mice immunized with recombinant 19-kDa protein, killed M. tuberculosis, or live bacillus Calmette Guerin infection. T cell lines specific for peptide 19.7 were of the CD4 phenotype. Significantly, sequence analysis revealed that residues 61 to 80 of the 19-kDa protein exhibited considerable homology with a single 20-amino acid sequence (residues 120 to 140), but not with any other region of the 28-kDa protein expressed in Mycobacterium leprae. This finding is the first evidence of epitope-restricted homology between otherwise structurally unrelated microbial Ag.     

Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide

Detergent-solubilized NADPH-cytochrome P-450 reductase was purified from porcine hepatic microsomes and compared to the rabbit enzyme isolated under identical conditions. The porcine enzyme had an equivalent specific activity toward cytochrome c compared to the rabbit enzyme. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the porcine enzyme exhibited a major band at Mr = 80,000 and two additional bands at Mr = 20,000 and 60,000. The 20-kDa fragment was shown to be the COOH-terminal portion of the protein which contains a hydrophobic sequence of 28 residues homologous to the pyrophosphate-binding portion of the FAD-binding protein p-hydroxybenzoate hydroxylase. The 60-kDa fragment corresponded to the NH2-terminal portion of the protein since this peptide and the intact protein have blocked NH2 terminal. The trypsin-solubilized porcine enzyme has an NH2-terminal sequence which is homologous to the equivalent trypsin-solubilized enzymes from rat and rabbit (80% sequence homology). Eight cysteine-containing peptides were isolated from a tryptic digest of the S-carboxymethylated pig enzyme. Significant sequence homology was not found between these peptides and other flavoproteins, except for one peptide (Glu-Val-Gly-Glu-Thr-Leu-Leu-Tyr-Tyr-Gly-Cys-Arg) which exhibited partial homology with the known NADPH-binding site of glutathione reductase. When the NADPH-protected enzyme was first S-alkylated with unlabeled iodoacetate, NADPH depleted, and further alkylated with 14C-labeled iodoacetate, the above radiolabeled peptide was isolated from a tryptic digest. The equivalent peptide was also isolated by a similar procedure from rabbit liver cytochrome P-450 reductase.     

Primary structure of a linker subunit of the tube worm 3000-kDa hemoglobin

The deep-sea tube worm Lamellibrachia contains two giant extracellular hemoglobins, a 3000-kDa hemoglobin and a 440-kDa hemoglobin. The former consists of four heme-containing chains (AI-AIV) and two linker chains (AV and AVI) for the assembly of the heme-containing chains. The 440-kDa hemoglobin consists of only four heme-containing chains (Suzuki, T., Takagi, T., and Ohta, S. (1988) Biochem. J. 255, 541-545). The complete amino acid sequence of a linker subunit (chain AV) has been determined by automated Edman sequencing of the peptides derived by digestions with lysyl endopeptidase and endoproteinase Asp-N. The chain is composed of 224 amino acid residues, and the molecular mass for the protein moiety was calculated to be 24,894 Da. An Asn-X-Thr sequence which is possible as a glycosylation site was suggested at positions 108-110. A computer-assisted homology search showed that the sequence shows no notable homology with any other globins and proteins. However a careful alignment of the linker sequence with a heme-containing chain sequence suggested that there is a slight, but significant homology between the two sequences. The alignment also suggested that the linker resulted from gene duplication of a heme-containing chain with a three exon-two intron structure, and that the first exon of domain 1 and the last exon of domain 2 had been lost during evolution. In our alignment, domain 1 has the heme-binding proximal histidine, but domain 2 does not. This is the first linker subunit to be sequenced completely.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

Primary structure of human plasma fibronectin. Characterization of a 31,000-dalton fragment from the COOH-terminal region containing a free sulfhydryl group and a fibrin-binding site

The 31-kDa domain of human plasma fibronectin has been completely characterized. This fragment is located at the COOH-terminal end of the molecule immediately preceding the 3-kDa interchain disulfide-containing peptide. The 31-kDa domain was obtained after trypsin digestion of fibronectin and purified by affinity chromatography on gelatin- and heparin-Sepharose columns. The fragment eluted in the heparin-unbound fraction and was further purified by DEAE-cellulose and high performance liquid chromatography. The 31-kDa fragment contained a fibrin-binding site (fibrin II site) which was only active at physiological NaCl concentrations and therefore differed from that located in the NH2-terminal domain which also bound at lower NaCl concentrations. The 31-kDa domain bound to thiopropyl-Sepharose and was shown to contain a free sulfhydryl group located at position 35 in the sequence. To determine the complete amino acid sequence of this fragment, a trypsin digestion was performed on the reduced and alkylated 31-kDa domain, and the 17 resulting peptides were isolated by high performance liquid chromatography; their amino acid compositions and amino acid sequences have been determined, and the arrangement of peptides was achieved by comparison with the sequences deduced from human and rat cDNA clones and with a related plasmic fragment from bovine fibronectin. Comparison of these three sequences showed 23 amino acid differences between human and rat fibronectin and 16 between human and bovine fibronectin. This represents a 91 and 94% homology, respectively. An interesting finding is that the 31-kDa fragment contains a deletion of 31 residues when compared to the rat cDNA sequence. This deletion appears to represent a species difference since it is due to a shorter mRNA in the case of human fibronectin.     

The properdin-like type I repeats of human thrombospondin contain a cell attachment site

Thrombospondin (TS) is a modular adhesive glycoprotein that contains three domains previously implicated in the attachment of cells to TS. These include the amino-terminal heparin-binding domain, the carboxy terminal cell or platelet-binding domain, and an RGDA sequence of TS. We have characterized a mAb against human TS, designated A4.1, which inhibits the attachment of human melanoma cells (G361) to TS. The epitope for A4.1 lies within the amino terminal half of the central stalklike region of TS which is distinct from the three known cell attachment sites. This region of TS is recovered in a 50-kD peptide after chymotryptic digestion of TS in EDTA. It contains the procollagen-like domain of TS as well as three type I repeats of a 60-residue segment homologous to two malarial proteins and the complement proteins properdin, and factors C6 through C9. The purified chymotryptic fragment is an effective attachment factor for G361 cells. A4.1 blocks adhesion to the 50-kD domain, as do some sulfated glycoconjugates. RGD (and RGE) peptides and mAbs against other domains of TS are not inhibitory. Peptides (19 mers) based on the core homology sequence of the three type I repeats of TS are potent attachment factors for these cells, and this adhesion is also inhibited by sulfated glycoconjugates. A polyclonal antibody raised against one of these peptides inhibits adhesion of G361 cells to the peptides, to the 50-kD fragment and to intact TS. Thus a new cell-adhesion site has been identified in TS whose sequence is very similar to the site identified in region II of the circumsporozoite protein of malaria parasites (Rich, K. A., F. W. George IV, J. L. Law, and W. J. Martin. 1990. Science (Wash. DC) 249:1574-1577. Thus there may be a common receptor which binds TS, malarial proteins, and properdin.     

Proteolytic processing of the mosquitocidal toxin from Bacillus sphaericus SSII-1.

The 97-kDa protein Mtx21, derived from the 100-kDa mosquitocidal protein (Mtx) from Bacillus sphaericus SSII-1 by the deletion of the putative signal sequence, was expressed as a fusion protein with glutathione S-transferase in Escherichia coli, and the fusion protein was purified by affinity chromatography. The fusion protein bound to glutathione agarose was cleaved with thrombin to release the Mtx21 protein. The 97-kDa Mtx21 protein was found to be toxic to Culex quinquefasciatus larvae with a 50% lethal concentration of 15 ng/ml. Treating Mtx21 with crude mosquito larval gut extracts gave rise to two major peptides of 70 and 27 kDa. Treating the 97-kDa Mtx21 protein with trysin also gave rise to a similar proteolytic cleavage pattern. N-terminal sequencing showed that the 27-kDa peptide was derived from the N-terminal region of the 97-kDa protein and that the 70-kDa protein was from the C-terminal region of the 97-kDa protein. The 27-kDa peptide has all the previously identified regions of homology with the catalytic peptides of the ADP-ribosyltransferase toxins, such as pertussis toxin S1 peptide, while the 70-kDa peptide has three internal regions of homology.