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J A Wells

Publications and source records attributed to J A Wells.

At least 19 recordsLinked to original sources
PubMed1992

Incorporation of a stabilizing Ca(2+)-binding loop into subtilisin BPN'.

A rational approach was taken to improve the stability of subtilisin BPN' to autoproteolysis. Two sites of autoproteolysis were identified by isolation of early autolysis products and amino-terminal sequence analysis. These studies showed that subtilisin rapidly cleaves Ala48-Ser49 and Ser163-Thr164 peptide bonds at elevated temperatures. These two sites appear in regions of high mobility as estimated from crystallographic B-factors and are in extended surface loops. To improve the resistance to thermal-induced autolysis, we replaced sequences around these two sites with sequences derived from a thermophilic homologue of subtilisin, thermitase. Thermitase contains a Ca(2+)-binding site in the region surrounding Ser49. When the Ca(2+)-binding segment of thermitase corresponding to residues 45-63 of subtilisin BPN' was installed into subtilisin BPN', the chimeric protein gained the ability to bind another Ca2+ with moderate affinity (Kd approximately 100 microM). This enzyme had the same kcat as wild-type, had a KM value 8-fold larger than wild-type, and was slightly less stable to thermal inactivation in EDTA. However, in 10 mM CaCl2, the mutant subtilisin BPN' was 10-fold more stable to irreversible inactivation at 60 degrees C than wild-type subtilisin BPN' as measured by residual activity against the substrate sAAPF-pna. Next, mutations and deletions derived from thermitase were introduced near the second autolysis loop in subtilisin BPN' (residues 158-165). However, all of these mutants were less stable than wild-type subtilisin. Thus, some (but not all) mutations derived from a thermophilic homologue near sites of autolysis can be stabilizing to a mesophilic protease.

Amino Acid Sequence↗
PubMed1992

High resolution functional analysis of antibody-antigen interactions.

A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from approximately 10(7) to 3 x 10(10) M-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbent assay were used to create high resolution "functional epitopes" for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 A radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg greater than Pro greater than Glu approximately Asp approximately Phe approximately Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.

Alanine↗
PubMed1992

Rational design of potent antagonists to the human growth hormone receptor.

A hybrid receptor was constructed that contained the extracellular binding domain of the human growth hormone (hGH) receptor linked to the transmembrane and intracellular domains of the murine granulocyte colony-stimulating factor receptor. Addition of hGH to a myeloid leukemia cell line (FDC-P1) that expressed the hybrid receptor caused proliferation of these cells. The mechanism for signal transduction of the hybrid receptor required dimerization because monoclonal antibodies to the hGH receptor were agonists whereas their monovalent fragments were not. Receptor dimerization occurs sequentially--a receptor binds to site 1 on hGH, and then a second receptor molecule binds to site 2 on hGH. On the basis of this sequential mechanism, which may occur in many other cytokine receptors, inactive hGH analogs were designed that were potent antagonists to hGH-induced cell proliferation. Such antagonists could be useful for treating clinical conditions of hGH excess, such as acromegaly.

Antibodies, Monoclonal↗
PubMed1992

Immunodominant structures of human growth hormone identified by homolog-scanning mutagenesis.

Homolog-scanning mutagenesis has been reported to be useful in elucidating the antigenic epitopes recognized by monoclonal antibodies and hGH binding to its receptor. However, little is known about which structures are recognized as immunodominant by murine serum antibodies. Therefore, the previously published series of hGH homologs and additional mutants of human placental lactogen (hPL), porcine growth hormone (pGH), and human prolactin (hPRL) were examined for their interaction with murine serum derived anti-hGH antibodies. As compared to wild-type hGH, nine of the nineteen segment substituted mutants tested showed a significant reduction in binding to anti-hGH sera. These disruptive substitutions mapped to 5 regions on a structural model of hGH: the length of helix 1 (residues 11-33), the loop between the first disulfide bond and helix 2 (residues 54-74), the beginning of helix 3 (residues 109-112), the carboxyl half of helix 4 (residues 167-182), and the final carboxyl terminus segment of the molecule (residues 184-191). In terms of the current structural model, three of the five immunodominant regions (the loop between residues 54-74, central portion of helix 4 to the carboxyl terminus and part of the amino terminus region of helix 1) closely overlaps the hGH receptor binding epitopes.

Amino Acid Sequence↗
PubMed1992

Rapid evolution of peptide and protein binding properties in vitro.

A significant bottleneck in protein engineering arises from the problem of identifying particular molecules with new functions from a potentially enormous range of peptide or protein variants. Two areas of emerging technology, phage display and multiple peptide synthesis, provide new means of screening huge libraries in vitro for novel binding properties. This review is also published in Current Opinion in Structural Biology 1992, 2:597-604.

Bacteriophages↗
PubMed1992

Effect of aging and exercise on GLUT-4 glucose transporters in muscle.

This study was conducted to investigate whether changes in muscle glucose transporter GLUT-4 protein might be associated with a previously reported deterioration in glucose tolerance with aging, and, furthermore, to determine whether exercise training could increase GLUT-4 protein levels in older animals. GLUT-4 protein concentration was measured in soleus, gastrocnemius, and extensor digitorum longus muscles of trained (10 or 15 wk treadmill running) and untrained young (6-8 mo), middle-aged (15-17 mo), and old (27-29 mo) Fischer 344 rats. All GLUT-4 protein values were expressed as a percent of the mean for the young untrained group. Two-way analysis of variance demonstrated significant main effects of both training and aging in the gastrocnemius and soleus muscles. Exercise training produced significant increases in GLUT-4 protein in the soleus muscle of young (273 +/- 32.9 vs. 100 +/- 38.5%) and middle-aged rats (215 +/- 19.9 vs. 108 +/- 33.2%) compared with sedentary controls. Similar significant increases were also found in the gastrocnemius muscle of young (169 +/- 20.1 vs. 100 +/- 5.8%) and middle-aged rats (167 +/- 46.7 vs. 60 +/- 7.9%) with training. In the oldest rats, GLUT-4 was not significantly increased with training, but the trend toward an increase was apparent in all three muscle types. The main effect of aging was primarily due to a statistically significant difference between the old trained and young trained rats. A trend toward decreased GLUT-4 with aging was apparent in the untrained animals, but this was not statistically significant.

Aging↗
PubMed1992

Growth hormone augments superoxide anion secretion of human neutrophils by binding to the prolactin receptor.

Recombinant human growth hormone (HuGH) and human prolactin (HuPRL), but not GH of bovine or porcine origin, prime human neutrophils for enhanced superoxide anion (O2-) secretion. Since HuGH, but not GH of other species, effectively binds to the HuPRL receptor (HuPRL-R), we used a group of HuGH variants created by site-directed mutagenesis to identify the receptor on human neutrophils responsible for HuGH priming. A monoclonal antibody (MAb) directed against the HuPRL-R completely abrogated O2- secretion by neutrophils incubated with either HuGH or HuPRL, whereas a MAb to the HuGH-R had no effect. The HuGH variant K172A/F176A, which has reduced affinity for both the HuGH-binding protein (BP) and the HuPRL-BP, was unable to prime human neutrophils. This indicates that priming is initiated by a ligand-receptor interaction, the affinity of which is near that defined for receptors for PRL and GH. Another HuGH variant, K168A/E174A, which has relatively low affinity for the HuPRL-BP but slightly increased affinity for the HuGH-BP, had much reduced ability to prime neutrophils. In contrast, HuGH variant E56D/R64M, which has a similar affinity as wild-type HuGH for the HuPRL-BP but a lower affinity for the HuGH-BP, primed neutrophils as effectively as the wild-type HuGH. Finally, binding of HuGH to the HuPRL-BP but not to the HuGH-BP has been shown to be zinc dependent, and priming of neutrophils by HuGH was also responsive to zinc. Collectively, these data directly couple the binding of HuGH to the HuPRL-R with one aspect of functional activation of human target cells.

Adult↗
PubMed1992

Prevalence of obesity: a comparative survey in France, the United Kingdom and the United States.

We investigated the differences in the prevalence of obesity between France, the United Kingdom and the United States in 1988. The analysis was made on a total sample of 5580 subjects, representative of the population aged 16-50 years in the three countries. The same questionnaire was used in all three countries. Body mass index (kg/m2) was used to assess corpulence. Significant differences in the prevalence of obesity were observed among the three countries: 7% of the population in France was obese, 9% in the UK and 15% in the USA (P less than 0.001). There was a strikingly high percentage of very obese women (more than 50% overweight) in the USA (8% of the population) as compared to the two European countries (2% in France and 3% in the UK). In all three countries, obesity was related to sex, age, level of education, marital status, physical exercise and smoking. An inverse association was found between obesity and alcohol consumption in the USA, but not in France nor in the UK. In men, prevalence of obesity remained significantly higher in the USA than in France or in the UK when adjusting for the obesity-related factors. In women, differences in prevalence of obesity between the three countries varied according to the level of exercise, income and alcohol consumption.

Adolescent↗
PubMed1991

Selecting high-affinity binding proteins by monovalent phage display.

Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.

Amino Acid Sequence↗
PubMed1991

Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule.

Human growth hormone (hGH) forms a 1:2 complex with the extracellular domain of its receptor-binding protein (hGHbp) as studied by crystallization, size exclusion chromatography, calorimetry, and a previously undescribed fluorescence quenching assay. These and other experiments with protein engineered variants of hGH have led to the identification of the binding determinants for two distinct but adjacent sites on hGH for the hGHbp, and the data indicated that there are two overlapping binding sites on the hGHbp for hGH. Furthermore, the binding of hGH to the hGHbp occurred sequentially; a first hGHbp molecule bound to site 1 on hGH and then a second hGHbp bound to site 2. Hormone-induced receptor dimerization is proposed to be relevant to the signal transduction mechanism for the hGH receptor and other related cytokine receptors.

Amino Acid Sequence↗
PubMed1991

Dimerization of human growth hormone by zinc.

Size-exclusion chromatography and sedimentation equilbrium studies demonstrated that zinc ion (Zn2+) induced the dimerization of human growth hormone (hGH). Scatchard analysis of 65Zn2+ binding to hGH showed that two Zn2+ ions associate per dimer of hGH in a cooperative fashion. Cobalt (II) can substitute for Zn2+ in the hormone dimer and gives a visible spectrum characteristic of cobalt coordinated in a tetrahedral fashion by oxygen- and nitrogen-containing ligands. Replacement of potential Zn2+ ligands (His18, His21, and Glu174) in hGH with alanine weakened both Zn2+ binding and hGH dimer formation. The Zn(2+)-hGH dimer was more stable than monomeric hGH to denaturation in guanidine-HCl. Formation of a Zn(2+)-hGH dimeric complex may be important for storage of hGH in secretory granules.

Amino Acid Sequence↗
PubMed1991

Probing the mechanism and improving the rate of substrate-assisted catalysis in subtilisin BPN'.

A mutant of the serine protease, subtilisin BPN', in which the catalytic His64 is replaced by Ala (H64A), is very specific for substrates containing a histidine, presumably by the substrate-bound histidine assisting in catalysis [Carter, P., & Wells, J.A. (1987) Science (Washington, D.C.) 237, 394-399]. Here we probe the catalytic mechanism of H64A subtilisin for cleaving His and non-His substrates. We show that the ratio of aminolysis to hydrolysis is the same for ester and amide substrates as catalyzed by the H64A subtilisin. This is consistent with formation of a common acyl-enzyme intermediate for H64A subtilisin, analogous to the mechanism of the wild-type enzyme. However, the catalytic efficiencies (kcat/KM) for amidase and esterase activities with His-containing substrates are reduced by 5000-fold and 14-fold, respectively, relative to wild-type subtilisin BPN, suggesting that acylation is more compromised than deacylation in the H64A mutant. High concentrations of imidazole are much less effective than His substrates in promoting hydrolysis by the H64A variant, suggesting that the His residue on the bound (not free) substrate is involved in catalysis. The reduction in catalytic efficiency kcat/KM for hydrolysis of the amide substrate upon replacement of the oxyanion stabilizing asparagine (N155G) is only 7-fold greater for wild-type than H64A subtilisin. In contrast, the reductions in kcat/KM upon replacement of the catalytic serine (S221A) or aspartate (D32A) are about 3000-fold greater for wild-type than H64A subtilisin, suggesting that the functional interactions between the Asp32 and Ser221 with the substrate histidine are more compromised in substrate-assisted catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗
PubMed1991

The importance of a distal hydrogen bonding group in stabilizing the transition state in subtilisin BPN'.

Stabilization of an oxyanion transition state is important to catalysis of peptide bond hydrolysis in all proteases. For subtilisin BPN', a bacterial serine protease, structural data suggest that two hydrogen bonds stabilize the tetrahedral-like oxyanion intermediate: one from the main chain NH of Ser221 and another from the side chain NH2 of Asn155. Molecular dynamic studies (Rao, S., N., Singh, U., C. Bush, P. A., and Kollman, P. A. (1987) Nature 328, 551-554) have indicated the gamma-hydroxyl of Thr220 may be a third hydrogen bond donor even though it is 4A away in the static x-ray structure. We have probed the role of Thr220 by replacing it with serine, cysteine, valine, or alanine by site-directed mutagenesis. These substitutions were intended to alter the size and hydrogen bonding ability of residue 220. Removal of the gamma-hydroxyl group reduced the transition state stabilization energy (delta delta GT) by 1.8-2.1 kcal/mol depending upon the substitution. By comparison, removal of the gamma-methyl group in the Thr220 to serine mutation only decreased delta GT by 0.5 kcal/mol. The gamma-hydroxyl of Thr220 is most important for catalysis, not substrate binding, because virtually all of the effects were on kcat and not KM. The role of the Thr220 hydroxyl is functionally independent from the amide NH2 of Asn155 because the free energy effects of double alanine mutants at these two positions are additive. These data indicate that a distal hydrogen bond donor, namely the hydroxyl of Thr220, plays a functionally important role in stabilizing the oxyanion transition state in subtilisin which is independent of Asn155.

Amino Acid Sequence↗
PubMed1991

Mutational analysis and protein engineering of receptor-binding determinants in human placental lactogen.

Human placental lactogen (hPL) shares 85% sequence identity to human growth hormone (hGH) yet has some very different receptor-binding properties. For example, hPL binds 2300-fold weaker than hGH to the hGH receptor, yet these two hormones have similar affinities for prolactin receptors. We have expressed hPL in Escherichia coli, and we show that, like hGH, hPL requires zinc for tight binding to the extracellular domain of the human prolactin receptor (hPRLbp). In fact, hPL contains virtually the same receptor-binding determinants and zinc ligands (His-18, His-21, and Glu-174) that hGH uses for coordinating zinc in the hGH.hPRLbp complex. As with hGH, mutation of Glu-174 to Ala in hPL reduces the affinity for the hPRLbp by 1400-fold. We can increase the affinity of hPL by over 200-fold for the hGHbp by installing four hGH receptor determinants that are not conserved in hPL. By simultaneously introducing E174A, we produced a pentamutant whose binding affinity for the hGHbp is only 1.6-fold weaker than hGH, but whose binding affinity for the hPRLbp is weaker by greater than 1000-fold relative to wild-type hPL. Thus, we have identified an hPRLbp epitope in hPL, "recruited" an hGHbp epitope into hPL, and produced receptor selective analogs of hPL that are designed to bind tightly to either, neither, or both receptors. Such variants should be important molecular probes to link specific receptor-binding, activation, and biological events.

Amino Acid Sequence↗
PubMed1991

A systematic mutational analysis of hormone-binding determinants in the human growth hormone receptor.

A mutational strategy is presented that allowed us to identify hormone-binding determinants in the extracellular portion of the human growth hormone receptor (hGHbp), a 238-residue protein with sequence homology to a number of cytokine receptors. By systematically replacing side chains with alanine we probed the importance of charged residues (49 total, typically located on the surface), aromatic residues (9 total), and neighbors of these (26 total). The alanine substitutions that were most disruptive to hormone binding are located predominantly in four segments of a cysteine-rich domain in the hGHbp, and collectively they form a patch when mapped upon a structural model proposed for cytokine receptors. Control experiments with monoclonal antibodies confirmed that most of these alanine substitutions do not disrupt the overall antigenic structure of the hGHbp. This high-resolution functional analysis will complement structural studies and provides a powerful basis for evaluating and engineering the energetics of hormone-receptor interactions. Moreover, the hormone-binding determinants identified here may be similarly located in other, homologous, receptors.

Alanine↗
PubMed1991

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution.

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗