Biomedical subjects

H B Lowman

Publications and source records attributed to H B Lowman.

27 records · Page 2Linked to original sources

PubMed1994

The stoichiometry of growth hormone-binding protein complexes in human plasma: comparison with cell surface receptors.

The recent demonstration of two independent receptor-binding sites (sites 1 and 2) on human GH (hGH) raises the question of the stoichiometry of circulating GH-binding protein (GH-BP) complexes in human plasma (i.e. is it one hGH per one GHBP or one hGH per two GHBPs?). Previous studies have all assumed 1:1 binding in plasma, based on gel exclusion chromatography and cross-linking data. To address this issue, human plasma was incubated with radioiodinated hGH as well as hGH mutants that had either a Tyr103-->Ala or a Gly120-->Arg substitution in the region of binding site 2. The former mutant retains normal site 2 binding activity even when iodinated; the latter has binding site 2 inactivated. Bound and free hGH were then separated on a Sephadex G-100 column according to a standard protocol for measuring GHBP. In all three cases, more than 90% of the high affinity GH-BP complex eluting from the column was consistent with 1:1 binding. Similar results were obtained when a physiological amount of recombinant or purified natural GHBP was substituted for plasma. However, at supraphysiological concentrations of GHBP, an additional component corresponding to the 2:1 complex eluted from the column; the relative proportions of the 2:1 and 1:1 complexes were dependent on the GHBP concentration. These data suggest that at physiological GHBP levels in plasma, the 1:1 complex predominates, and that small amounts of the 2:1 complex may be difficult to detect because of partial peak overlap with the 1:1 complex, dissociation, and, in whole plasma, coelution with the low affinity GHBP complex. Calculation of the theoretical partition of hGH between 1:1 and 2:1 complexes indicated that at concentrations of GHBP prevailing in plasma (approximately 1 nmol/L), the 1:1 complex predominates, but that at the high receptor concentrations prevailing at the cell surface (60 nmol/L to 6.7 mumol/L, depending on the cell type), virtually all hGH is captured in a 2:1 complex. These findings are consistent with the present and previous experimental data on the size of the circulating high affinity GH-BP complex, as well as with those indicating the importance of GH-induced receptor dimerization for GH action. A functional consequence of the large concentration difference between GHBP in plasma and GH receptors at the cell surface is that the circulating GHBP can serve as a dynamic buffer, modulating bound and free GH and prolonging its half-life, whereas the receptor acts as a dominant force in unidirectional capture of GH.(ABSTRACT TRUNCATED AT 400 WORDS)

Carrier Proteins↗

PubMed1993

Affinity maturation of human growth hormone by monovalent phage display.

We describe a selection procedure for construction of very high affinity variants of human growth hormone (hGH) for binding to the extra cellular domain of its receptor (called the hGHbp). Five different libraries of mutated hGH genes (each containing approximately 2 x 10(5) protein variants) were created by randomly mutating four different codons at residues that were shown to be important for receptor binding by structural or functional criteria. Mutated proteins were displayed as single copies from their respective filamentous phagemid particles and sorted in vitro for binding to the immobilized hGHbp. Phagemid particles that bound the immobilized hGHbp were eluted and propagated. After three to seven rounds of binding enrichments, hGH variants were isolated that contained 2 to 4 mutations and exhibited three- to sixfold improvements in binding affinity. Because of the limits of DNA transfection efficiency in creating the library we could not sample thoroughly mutations at more than four codons at once. Nonetheless, the free energy effects for these mutations acted cumulatively. Thus, by combining affinity enhanced mutants from libraries independently sorted we created an hGH variant with 15 substitutions that bound approximately 400-fold more tightly to the hGHbp than wild-type hGH. The affinity enhancements occurred predominantly by slowing the off-rate of the hormone (> 60-fold), and partly through increasing the on-rate (up to 4-fold). Residues that were shown to be important for binding by alanine-scanning were most highly conserved after binding selection, and interestingly many of them could be further improved. Thus, we found it most effective to randomly mutate the residues that were shown to modulate affinity by alanine-scanning, and to combine the selectants from separate libraries that exhibit the highest affinities. The selection procedure and mutagenesis strategy provides a framework for affinity maturation of protein-protein complexes.

Amino Acid Sequence↗

PubMed1993

The molecular basis for growth hormone-receptor interactions.

High-resolution mutational and structural analyses of purified components have revealed a great deal about the molecular basis for growth hormone action. The structural and functional aspects of the interactions between hGH and its receptors have been largely elaborated. From these studies it has been possible to engineer homologues of hGH to bind to the hGH receptor and act as potential antagonists. Receptor-selective and high-affinity analogs have also been constructed based on a combination of alanine scanning and monovalent phage display. From this molecular work much has been revealed about the biology of hGH (Fig.9). Our data suggest that hGH is stored in the pituitary as a (Zn2+,hGH)2 complex. On release from somatotropic vesicles it dissociates into a monomeric form and reveals its primary receptor binding site (site 1). Free hGH can bind to the hGHbp in serum to form monomeric or dimeric complexes that slow the clearance of hGH (Moore et al., 1989). However, because the affinity for the full-length receptor is greater, hGH can bind to it preferentially. Furthermore, the constitutive levels of the hGHbp (approximately 0.5 to 1 nM) (Baumann et al., 1986; Herrington et al., 1986) are considerably below the levels of hGH after pulsatile release (approximately 2 to 5 nM) (Thompson et al., 1972). Our data indicate that hGH binds to the hGH receptor on cell membranes through site 1 and subsequently forms dimers through site 2. We believe a similar process may occur for hGH to activate the hPRL receptor, except that Zn2+ is required for site 1 association. Such receptor dimers are then activated and capable of interacting with other cellular components that may mediate the hGH "signal." Recently, based upon this proposed mechanism, we produced potent antagonists to the hGH receptor (Fuh et al., 1992) and hPRL receptor (G. Fuh, P. Colosi, W. Wood, and J. Wells, unpublished results). These antagonists bind tightly to site 1 but are blocked in their ability to bind site 2 and dimerize the receptor. We believe these methods and discoveries will be relevant to the study of signaling by other hematopoietic hormones and receptors as well as other hormones and receptors.

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↗

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

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↗

PubMed1990

Temperature-mediated regulation and downstream inducible selection for controlling gene expression from the bacteriophage lambda pL promoter.

We have examined in detail the effects of various induction temperatures on the expression of a heterologous fusion gene controlled by the bacteriophage lambda PL promoter in a heat-inducible Escherichia coli expression system which utilizes the CIts857 repressor. Experiments performed over a temperature range spanning 29-42 degrees C indicate that, under our conditions, temperatures as low as 29 degrees C may be required to fully repress the CI857-controlled transcription from PL, and that the highest protein yields are obtained after induction at 36 degrees C for 6 h. We cloned the cat reporter gene downstream from a heterologous gene controlled by PL and found that cat expression at a low induction temperature permits the monitoring of productive transcription through the heterologous gene and thus aids in selecting transformants that are capable of producing the heterologous protein in E. coli.

Bacteriophage lambda↗

PubMed1988

High-level expression of the simian virus 40 genes LP1, VP1 and VP2 as fusion proteins in Escherichia coli.

The complete sequences of the SV40 agnogene (LP1) and the genes coding for the capsid proteins VP1 and VP2 have been cloned into Escherichia coli expression plasmids. High levels of expression were obtained when the SV40 genes were inserted into the coding sequence of the influenza virus NS1 gene, which has previously been expressed in E. coli. The NS1A-LP1 and NS1A-VP2 chimeric proteins consist of the 81 N-terminal residues of NS1 (designated as peptide NS1A) fused to the complete sequence of the corresponding SV40 protein. The NS1A-VP1 chimera consists of NS1A followed by a linker of nine arbitrary residues and the complete sequence of the SV40 major capsid protein. The observed levels of expression vary considerably among the three chimeric proteins, ranging from approx. 70 micrograms/ml in the case of NS1A-LP1 to approx. 5 micrograms/ml in the case of NS1A-VP2. Cyanogen bromide cleavage of the NS1A-LP1 fusion protein produces fragments with Mrs expected for isolated NS1A and LP1 peptides. A plasmid has also been constructed which expresses the NS1A peptide in high yield.

Amino Acid Sequence↗

PubMed1986

On the recognition of helical RNA by cobra venom V1 nuclease.

The V1 nuclease from cobra venom preferentially hydrolyzes double helical RNA and has been used extensively for detecting RNA secondary structure. To increase the utility of this enzyme as an RNA structure probe, we have investigated its properties and substrate specificity, using assays for polynucleotide hydrolysis based on fluorescent polynucleotide derivatives. Enzymatic activity requires both Na+ and Mg2+, with optima at 100 and 0.3 mM, respectively. From the sharp decrease in enzyme activity above 100 mM Na+ we estimate that 3-4 ionic interactions between the protein and polynucleotide phosphates take place. Analysis of products remaining after extensive V1 digestion also shows that the minimum size substrate is 4-6 nucleotides long. Helical RNAs and DNAs have Michaelis constants a factor of 3-10 times lower than most single-stranded RNAs. However, poly(epsilon A) has a Michaelis constant equal to the best synthetic double helices tested and is hydrolyzed at a rate comparable to helical RNA. The major V1 cutting sites in yeast tRNAPhe have Michaelis constants lower than any synthetic polymers. These data suggest that V1 nuclease recognizes any 4-6-nucleotide segment of polynucleotide backbone with an approximately helical conformation, but does not require that the bases be paired in a helix. A few single-stranded V1 cleavage sites are known in tRNA and rRNA, and their structures are consistent with the suggested V1 recognition site.

Base Sequence↗