Molecular and biochemical approaches to structure--function analysis of transforming growth factor alpha.
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Amino Acid Sequence↗
Biomedical subjects
J Y Tai
Publications and source records attributed to J Y Tai.
Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.
TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.
ADP Ribose Transferases↗
Substitution of lysine for arginine at position 42 of human transforming growth factor-alpha eliminates biological activity without changing internal disulfide bonds.
Transforming growth factor-alpha (TGF-alpha) is a growth-promoting protein that binds to the epidermal growth factor (EGF) receptor. To identify critical residues that govern TGF-alpha-EGF receptor binding, we prepared site-specific substitution mutants of TGF-alpha. Mutant proteins were tested in receptor-binding and mitogenesis assays. Semiconservative substitutions at positions 4, 12, 18, and 45 decreased biological activity 2.1- to 14-fold. The conservative substitution of lysine for arginine at position 42 completely eliminated biological activity. Amino acid composition analysis of proteolytic fragments from TGF-alpha and the Lys-42 mutant indicated that these proteins contained the same disulfide bonds. These studies suggest that arginine 42 may be a contact point for TGF-alpha-EGF receptor interaction.
Amino Acid Sequence↗
Structure-function analysis of synthetic and recombinant derivatives of transforming growth factor alpha.
Transforming growth factor alpha (TGF-alpha) is a 50-amino-acid peptide that stimulates cell proliferation via binding to cell surface receptors. To identify the structural features of TGF-alpha that govern receptor-ligand interactions, we prepared synthetic peptide fragments and recombinant mutant proteins of TGF-alpha. These TGF-alpha derivatives were tested in receptor binding and mitogenesis assays. Synthetic peptides representing the N terminus, the C terminus, or the individual disulfide constrained rings of TGF-alpha did not exhibit receptor-binding or mitogenic activity. Replacement of the cysteines with alanines at positions 8 and 21, 16 and 32, and 34 and 43 or at positions 8 and 21 and 34 and 43 yielded inactive mutant proteins. However, mutant proteins containing substitutions or deletions in the N-terminal region retained significant biologic activity. Conservative amino acid changes at residue 29 or 38 or both and a nonconservative amino acid change at residue 12 had little effect on binding or mitogenesis. However, nonconservative amino acid changes at residues 15, 38, and 47 produced dramatic decreases in receptor binding (23- to 71-fold) and mitogenic activity (38- to 125-fold). These studies indicate that at least three distinct regions of TGF-alpha contribute to biologic activity.
Amino Acid Sequence↗
Haemophilus influenzae type b polysaccharide-protein conjugate vaccine.
An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.
Animals↗
Molecular cloning of the temperature-sensitive 371 Kirsten murine sarcoma virus and expression in Escherichia coli of the mutant and wild-type viral Kirsten ras p21 proteins.
Rodent fibroblasts infected with the ts371 Kirsten murine sarcoma virus (KiMuSV) are temperature sensitive for the maintenance of transformation because of the production of an abnormal p21 protein. We cloned the ts371 KiMuSV provirus from the genome of a conditionally transformed nonproducer cell line, ts371 KiMuSV NRK clone 5 (T. Y. Shih, M. O. Weeks, H. A. Young, and E. M. Scolnick, J. Virol. 31:546-556, 1979). The molecularly cloned virus had 1,000-fold lower transformed focus-forming activity at 39 degrees C than at 34 degrees C. The ts371-v-Ki-ras gene differed from the wild type (wt) by a single point mutation, resulting in the substitution of arginine for glutamine at amino acid residue 43 of the encoded p21. A second difference from the published sequence for wt v-Ki-ras (N. Tsuchida, T. Ryder, and E. Ohtsubo, Science 217:937-939, 1982) at amino acid residue 37 was found. However, on sequencing the wt v-Ki-ras in this region, we found that it also contained a glutamate at residue 37. Preliminary characterization of bacterially expressed wt and ts371-v-Ki-ras p21 proteins is discussed.
Amino Acid Sequence↗
Isolation of type-specific polysaccharide antigen from group B type Ib streptococci.
Group B streptococcus type Ib (strain H36B) was subjected to digestion with extracellular muralytic enzymes prepared from Streptomyces albus. Type Ib-specific polysaccharide antigen was isolated from the lysate by alcohol precipitation and Sepharose 6B chromatography. The purified type Ib antigen has a Kd value of 0.31 on a Sepharose 4B column and contains four sugars, galactose, glucose, N-acetyl glucosamine, and sialic acid in a molar ratio of 2.05:0.86:1.00:0.90. Acid treatment (pH 2.0) of this polysaccharide results in partial degradation of the antigen (Kd = 0.41 on Sepharose 4B) with the loss of 93% of the sialic acid. The molar ratio of the remaining sugars in the polysaccharide remains identical to that in the native one. This suggests that the sialic acid is at the terminal position in the molecule. Both intact and acid-treated antigen cross-react with some type Ia and type Ic antisera as a result of the common Iabc determinant, but not with type II and type III antisera. Absorption studies indicate that Ib-specific determinant and Iabc determinant are on the same molecule and that sialic acid is not the cross-reactive determinant.
Antigens, Bacterial↗
Studies on Limulus lysate coagulating system.
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Amino Acids↗
The type-specific polysaccharides of Streptococcus suis.
Streptococcus suis types 1 and 2 were subjected to digestion with lysozyme. Serologically type-specific capsular polysaccharides were isolated from the lysates by ethanol precipitation followed by Sepharose 6B chromatography. The purified type 1 polysaccharide has a Kd value of 0.074 on a Sepharose 4B column and contains galactose, glucose, N-acetyl glucosamine, N-acetyl galactosamine, and sialic acid in a molar ratio of 2.42:1.00:1.00:1.13:1.39. The type 2 polysaccharide has a Kd value of 0.185 and is composed of rhamnose, galactose, glucose, N-acetyl glucosamine, and sialic acid in a molar ratio of 1.07:3.17:1.00:0.94:1.00. A comparison is drawn between the type polysaccharides of S. suis and those of group B streptococci.
Carbohydrates↗
Studies on Limulus amoebocyte lysate II. Purification of the coagulogen and the mechanism of clotting.
A coagulogen in Limulus lysate has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The purified coagulogen identified by its ability to clot with either trypsin or the purified Limulus clotting enzyme (Tai, J.Y., and Liu, T. Y. (1976) Fed. Proc. 35, 1486) has a molecular weight of 24,500. It consists of a single polypeptide chain of about 220 amino acids with glycine and serine as its NH2- and COOH-terminal residues; respectively. When acted upon by the Limulus clotting enzyme, the coagulogen releases a soluble C-peptide of about 45 amino acids and an insoluble coagulin of about 170 amino acids. The latter interacts in a noncovalent fashion to form the clot. Amino acid analyses together with the results of NH2- and COOH-terminal analyses suggest that the clot formation involves the cleavage of an--Arg--Lys--bond. Trypsin acts on the coagulogen to cause clotting by splitting the same peptide bond.
Amino Acids↗
Studies on Limulus amoebocyte lysate. Isolation of pro-clotting enzyme.
A pro-clotting enzyme capable of causing the gelation of clottable proteins in Limulus polyphemus (horseshoe crab) has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The activation of the pro-clotting enzyme depended on the presence of both Ca+ and endotoxin. It contained gamma-carboxyglutamic acids and gave a single NH2-terminal lysine. The enzyme was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and soy bean trypsin inhibitor, indicating that it is a serine protease. The molecular weight of the proclotting enzyme was determined to be at least 150,000 by sodium dodecyl sulfate-gel electrophoresis under reducing and denaturing conditions. The protein appears to consist of a single peptide chain, since exposure of the reduced and carboxymethylated enzyme to 6 M guanidine hydrochloride failed to dissociate it into any subunits.
Amino Acids↗
Primary structure of streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.
The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.
Amino Acid Sequence↗
Primary structure of streptococcal proteinase. III. Isolation of cyanogen bromide peptides: complete covalent structure of the polypeptide chain.
The following sequence has been derived for streptococcal proteinase. (See article). The sequence permits the assignment of the single cysteine residue essential for catalytic action at position 47 from the NH2 terminus of the protein. The tryptophan residue at the binding site of the enzyme is at position 214. A histidine residue at position 195 has been assigned as the catalytically important entity in the molecule. Streptococcal proteinase and papain, an enzyme with similar properties, are compared with respect to structure and function.
Amino Acid Sequence↗