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PubMed2001

Synthesis and catalytic antibody functionalization of dendrimers.

Antibody 38C2 catalyzed a retro-aldol process upon dendritic modified aliphatic polyesters. This catalytic system was studied in detail and displayed rate enhancements, k(cat)/k(uncat), of greater than 10(6). These antibody-catalyzed reactions took place in a stepwise manner yielding partially modified aldol-dendrimers until a fully substituted aldehyde dendrimer was formed. The catalytic antibody 38C2 only reacted with surface-exposed aldol moieties and did not significantly interact with the core groups for dendrons 4 and 8. For a higher generation dendron 8 the rate of unmasking slightly decreased presumably due to steric crowding of the aldol functionalities. In addition, catalytic antibody 38C2 was able to selectively differentiate block-hybrid dendrons and was regiospecific in the retro-aldol reaction of dendron 21. This is an inaugural report of a catalytic antibody utilizing dendrimers as substrates and suggests that antibodies could be used as selective catalysts for the controlled release and activation of specific molecules attached to biodegradable polymeric materials. Furthermore, this is the first example of catalytic antibody 38C2 displaying regioselectivity on a multifunctional aldol substrate. Important for synthetic applications is the antibody's ability to selectively differentiate regions on dendritic substrates and produce partly aldol functionalized dendrons under conditions mild enough to avoid beta-elimination.

Antibodies, Catalytic↗
PubMed1993

Differentiation between selected pairs of tripeptide diastereomers by tandem mass spectrometry on a hybrid tandem mass spectrometer.

Low-energy collisionally activated decomposition (CAD) and unimolecular decomposition of the [M+H]+ ions for X-L-Pro-L-Phe, where X is L-Ala, D-Ala, L-Asp, or D-Asp, allow easy differentiation between the LLL and DLL diastereomers. Tandem mass spectrometric (MS/MS) studies of the [M+H]+ ions formed by fast-atom bombardment (FAB) at various ion kinetic energies (Elab values) on a hybrid tandem instrument produced ions of different intensities for the diastereomers. The ratio of NH3 to H2O loss is 0.3 for the L-Ala peptide but 1.7 for the D-Ala isomer at 5 eV. In some L-Ala spectra, the [M+H-NH3]+ ion does not appear at all. The y2 ion is up to twice as abundant in the L-Ala spectra as in the D-Ala, while the b2 ion is somewhat more abundant for CAD of the D-Ala peptide for most collision energies investigated. The D-Asp peptide produces a b2 ion that is more than half-again as abundant as in the case of the L-Asp isomer, and an [M+H-H2O]+ ion that is up to twice as abundant in the D-Asp CAD spectra as in those of the L-Asp. The y1, a2, and phenylalanine immonium ions are each up to twice as abundant in the L-Asp spectra as in those of the D-Asp isomer. The major differences are correlated with force-field calculations on hydrogen-bonded tautomers.

Amino Acid Sequence↗
PubMed1988

Failure of Ro 15-4513 to antagonize ethanol in rat lines selected for differential sensitivity to ethanol and in Wistar rats.

An imidazobenzodiazepine, Ro 15-4513, acting as a partial inverse agonist at the central benzodiazepine receptors has been recently reported to reverse efficiently the intoxicating effects of ethanol. In studies designed to delineate the role of benzodiazepine receptors in the ethanol-induced motor impairment difference between two rat lines selectively bred for high and low sensitivity to ethanol, however, we could not antagonize the effects of ethanol by Ro 15-4513 in the tilting plane and horizontal wire tests. Neither could we observe any consistent antagonism of ethanol actions in Han:Wistar rats, although we used a wide range of Ro 15-4513 doses, injected the drug intraperitoneally or intragastrically and before or after ethanol administration, and carried out the tests for motor impairment (rotarod, horizontal wire test and intoxication rating) at various times after the drug administration. The ex vivo assay of flunitrazepam binding in brain homogenates revealed the presence of compound(s) inhibiting the binding after administration of Ro 15-4513. Ro 15-4513 antagonized the motor impairing effects of lorazepam. In conclusion, Ro 15-4513 failed to function as a specific antagonist of moderate doses of ethanol in several tests for motor impairment in different rat lines.

Animals↗
PubMed2001

T cell selection and differential activation on structurally related HLA-DR4 ligands.

Plasticity of TCR interactions during CD4(+) T cell activation by an MHC-peptide complex accommodates variation in the peptide or MHC contact sites in which recognition of an altered ligand by the T cell can modify the T cell response. To explore the contribution of this form of TCR cross-recognition in the context of T cell selection on disease-associated HLA molecules, we have analyzed the relationship between TCR recognition of the DRB1*0401- and DRB1*0404-encoded HLA class II molecules associated with rheumatoid arthritis. Thymic reaggregation cultures demonstrated that CD4(+) T cells selected on either DRB1*0401 or DRB1*0404 could be subsequently activated by the other MHC molecule. Using HLA tetramer technology we identify hemagglutinin residue 307-319-specific T cells restricted by DRB1*0401, but activated by hemagglutinin residues 307-319, in the context of DRB1*0404. One such clone exhibits an altered cytokine profile upon activation with the alternative MHC ligand. This altered phenotype persists when both class II molecules are present. These findings directly demonstrate that T cells selected on an MHC class II molecule carry the potential for activation on altered self ligands when encountering Ags presented on a related class II molecule. In individuals heterozygous for these alleles the possibility of TCR cross-recognition could lead to an aberrant immune response.

Animals↗
PubMed2003

Glutamate transport alteration triggers differentiation-state selective oxidative death of cultured astrocytes: a mechanism different from excitotoxicity depending on intracellular GSH contents.

Recent evidence has been provided for astrocyte degeneration in experimental models of neurodegenerative insults associated with glutamate transport alteration. To determine whether astrocyte death can directly result from altered glutamate transport, we here investigated the effects of L-trans-pyrrolidine-2,4-dicarboxylate (PDC) on undifferentiated or differentiated cultured rat striatal astrocytes. PDC induced death of differentiated astrocytes without affecting undifferentiated astrocyte viability. Death of differentiated astrocytes was also triggered by another substrate inhibitor but not by blockers of glutamate transporters. The PDC-induced death was delayed and apoptotic, and death rate was dose and treatment duration-dependent. Although preceded by extracellular glutamate increase, this death was not mediated through glutamate receptor stimulation, as antagonists did not provide protection. It involves oxidative stress, as a decrease in glutathione contents and a dramatic raise in reactive oxygen species preceded cell loss, and as protection was provided by antioxidants. PDC induced a similar percentage of GSH depletion in the undifferentiated astrocytes, but only a slight increase in reactive oxygen species. Interestingly, undifferentiated astrocytes exhibited twofold higher basal GSH content compared with the differentiated ones, and depleting their GSH content was found to render them susceptible to PDC. Altogether, these data demonstrate that basal GSH content is a critical factor of astrocyte vulnerability to glutamate transport alteration with possible insights onto concurrent death of astrocytes and gliosis in neurodegenerative insults.

Animals↗
PubMed1998

Differential thymic selection outcomes stimulated by focal structural alteration in peptide/major histocompatibility complex ligands.

The T lineage repertoire is shaped by T cell receptor (TCR)-dependent positive and negative thymic selection processes. Using TCR-transgenic (N15tg) beta2-microglobulin-deficient (beta2m-/-) RAG-2(-/-) H-2(b) mice specific for the VSV8 (RGYVYQGL) octapeptide bound to Kb, we identified a single weak agonist peptide variant V4L (L4) inducing phenotypic and functional T cell maturation. The cognate VSV8 peptide, in contrast, triggers negative selection. The crystal structure of L4/Kb was determined and refined to 2.1 A for comparison with the VSV8/Kb structure at similar resolution. Aside from changes on the p4 side chain of L4 and the resulting alteration of the exposed Kb Lys-66 side chain, these two structures are essentially identical. Hence, a given TCR recognizes subtle distinctions between highly related ligands, resulting in dramatically different selection outcomes. Based on these finding and the recent structural elucidation of the N15-VSV8/Kb complex, moreover, it appears that the germ-line Valpha repertoire contributes in a significant way to positive selection.

Amino Acid Sequence↗
PubMed2005

Maternal genetic effects on adaptive divergence between anadromous and resident brook charr during early life history.

The importance of directional selection relative to neutral evolution may be determined by comparing quantitative genetic variation in phenotype (Q(ST)) to variation at neutral molecular markers (F(ST)). Quantitative divergence between salmonid life history types is often considerable, but ontogenetic changes in the significance of major sources of genetic variance during post-hatch development suggest that selective differentiation varies by developmental stage. In this study, we tested the hypothesis that maternal genetic differentiation between anadromous and resident brook charr (Salvelinus fontinalis Mitchill) populations for early quantitative traits (embryonic size/growth, survival, egg number and developmental time) would be greater than neutral genetic differentiation, but that the maternal genetic basis for differentiation would be higher for pre-resorption traits than post-resorption traits. Quantitative genetic divergence between anadromous (seawater migratory) and resident Laval River (Québec) brook charr based on maternal genetic variance was high (Q(ST) > 0.4) for embryonic length, yolk sac volume, embryonic growth rate and time to first response to feeding relative to neutral genetic differentiation [F(ST) = 0.153 (0.071-0.214)], with anadromous females having positive genetic coefficients for all of the above characters. However, Q(ST) was essentially zero for all traits post-resorption of the yolk sac. Our results indicate that the observed divergence between resident and anadromous brook charr has been driven by directional selection, and may therefore be adaptive. Moreover, they provide among the first evidence that the relative importance of selective differentiation may be highly context-specific, and varies by genetic contributions to phenotype by parental sex at specific points in offspring ontogeny. This in turn suggests that interpretations of Q(ST)-F(ST) comparisons may be improved by considering the structure of quantitative genetic architecture by age category and the sex of the parent used in estimation.

Adaptation, Physiological↗
PubMed2006

Apolipoprotein E exerts selective and differential control over vitamin E concentrations in different areas of mammalian brain.

Apolipoprotein E (apoE) is known to be a risk factor for the incidence of Alzheimer's disease (AD). In addition, vitamin E has been reported to have a role in the treatment of AD. We examined the potential interrelationship between vitamin E and apoE in brain. As the first step, we determined the concentrations of alpha-tocopherol in selected brain regions of apoE-deficient mice at different ages. The mice were fed normal rodent chow. All regions of the brain in apoE-deficient mice contained less alpha-tocopherol than control samples at 2.5 months of age, the initial time of study. This trend continued for 9.5 months for most regions except the spinal cord and cerebellum. Tocopherol levels in these latter regions of apoE-deficient animals increased to control levels during the study. Serum alpha-tocopherol and cholesterol levels were high in the apoE-deficient animals; however, the CNS cholesterol levels were the same in apoE-deficient and control mice. This suggests that 1) the decline in brain alpha-tocopherol in apoE deficiency is not due to overall alterations in lipid metabolism; and 2) the processing of alpha-tocopherol in brain follows a separate pathway than that of cholesterol. Subcellular concentrations of alpha-tocopherol were unaltered by apoE deficiency indicating that intracellular handling of tocopherol is not affected by apoE. ApoE may be an important protein controlling vitamin E levels in specific brain regions. Further understanding of the interactions between apoE and vitamin E could be important in the appropriate use of vitamin E in AD.

Aging↗
PubMed2006

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

Animals↗
PubMed1997

Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors.

Previously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.

Adenoma↗
PubMed1983

Fluorogenic selective and differential medium for isolation of fecal streptococci.

Of 44 fluorogenic substrates tested for their ability to differentiate species of fecal streptococci, four yielded species-differentiating reactions. The remaining substrates either yielded uniformly positive, negative, or variable strain-dependent reactions. One substrate, 4-methylumbelliferone-alpha-D-galactoside, was hydrolyzed by Streptococcus bovis and S. faecium and its biotypes. 4-Methylumbelliferone-alpha-D-galactoside and a colorimetric starch substrate were incorporated into the fecal streptococcal selective medium of Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978). Three phenotypic groups were identifiable on the new fluorescent gentamicin-thallous-carbonate agar: (i) starch hydrolysis and fluorescence (S. bovis), (ii) no starch hydrolysis but fluorescence (S. faecium and its biotypes), and (iii) no starch hydrolysis or fluorescence (S. faecalis, S. avium, S. equinus, S. mitis, and S. salivarius). Of the presumptive identifications from sewage, swine, and bovine samples, 86% were confirmed as being correct. The new medium has potential application in water, food, environmental, and possibly clinical microbiology.

Animals↗
PubMed1977

The effects of genotype frequency and population density on fitness differentials in Escherichia coli.

Two strains of Escherichia coli K-12, a lac+ wild type and a lac- auxotroph, were grown both as pure and mixed cultures, using a serial transfer procedure. Four different growth media were employed, consisting of the same minimal salts solution, but different total concentrations of the sugars lactose, arabinose, and glucose (in proportions 5:4:1). Population densities and genotypic frequencies were assayed every 48 hours, at the time of transfer. Population density of the pure lac+ culture was greater than that of the pure lac- culture for all media; this was expected, since the latter cannot utilize lactose. Mixed cultures quickly approached the same density as the corresponding lac+ controls, and the frequency of the lac+ genotype increased steadily for all media. Trajectories of lambda = log (P divided by Q) were strictly nonlinear, indicating a dependence of the selective differential on population density and genotypic frequency. The rate of substitution decreased slightly with increasing sugar concentration, contrary to theoretical expectation. It was speculated that either the generation interval was longer for denser cultures (higher substrate concentrations) of that buildup of organic by-products reduced the selective differential in denser cultures. For a single medium, however, the behavior of completing genotypic strains was reasonably well predicted by theoretical models of frequency and density-dependent selection, the parameters of which may be related to the experimental inputs.

Culture Media↗
PubMed1999

Transmembrane tumor necrosis factor (TNF)-alpha inhibits adipocyte differentiation by selectively activating TNF receptor 1.

Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with multiple biological activities and exists in two forms as follows: a 17-kDa soluble form that is a cleaved product of the 26-kDa transmembrane form (mTNFalpha). It has been suggested that the transmembrane form of TNFalpha is mainly responsible for localized responses via cell-cell contact. Here, we have examined the activities of transmembrane TNFalpha in cultured adipocytes. A non-cleavable transmembrane form of TNFalpha (mTNFDelta1-9K11E) was expressed in several preadipocyte cell lines using retroviral gene transfer. In wild type preadipocytes carrying both TNF receptors, expression of mTNFDelta1-9K11E resulted in inhibition of the differentiation program. The extent of this varied depending on the nature and strength of the adipogenic stimuli. The TNF receptor responsible for this function was determined by expressing mTNFDelta1-9K11E in preadipocyte cell lines lacking either TNF receptor 1 (TNFR1), 2 (TNFR2), or both. In order to confirm the results in the same cellular background, TNF receptors were also reconstituted in the cell lines lacking corresponding receptors. These experiments demonstrated that TNFR1 was necessary and sufficient for mediating mTNFDelta1-9K11E-induced inhibition of adipogenesis and that this action was similar to that of soluble TNFalpha. In conclusion, our results indicate that mTNFDelta1-9K11E is biologically active in cultured adipocytes and can alter the adipogenic program of these cells by selectively activating TNFR1. This may have physiological implications where local TNFalpha actions are thought to be generated at sites such as adipose tissue.

Adipocytes↗
PubMed1984

Histologic differentiation of human fetal pancreatic explants transplanted into nude mice.

The transplantation of human fetal pancreas has been suggested as a means of treatment of insulin-dependent diabetes in man. We have obtained human fetal pancreata during the second trimester of pregnancy and transplanted 1-mm3 explants subcutaneously (s.c.) into both diabetic and nondiabetic nude mice, some of the tissue being cultured in vitro before implantation. These implants coalesced and grew. They were removed at intervals up to 37 wk later and showed selective differentiation of endocrine tissue that normally occurs in the fetus and neonate, with formation of bipolar, mantle, and mature islets. There was growth of this endocrine tissue with significantly more islets than in the freshly stained fetal pancreas assuming an average dimension larger than 150 micron, which is the reported mean diameter of a neonatal islet. Duct and fibrous tissue remained viable, but there was no definitive acinar tissue seen. The pancreata uncultured before implantation reached a larger size than that attained by those implants cultured before being transplanted, the difference probably being the amount of ductular and mesenchymal tissue still present. Of those glands cultured before transplantation, the longer the period of culture, the smaller the size the implants reached. Culture beyond 3 wk in vitro made it difficult to macroscopically locate the implant. These data show that, in human fetal pancreas removed from its usual environment, both selective differentiation of the endocrine component and growth of the islets can occur.

Animals↗
PubMed2002

Extracellular signal-regulated kinases and g protein-coupled receptors in megakaryocytic human erythroleukemia cells: selective activation, differential regulation, and dissociation from mitogenesis.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are a group of kinases that play an important role in proliferation and differentiation. In megakaryocyte-like human erythroleukemia (HEL) cells, ERK2 was found to be predominantly expressed and strongly activated by prostaglandin (PG) E(2), thrombin, and epinephrine. On the other hand, adenosine, ADP, ATP, and UTP did not significantly increase ERK1/2 phosphorylation. However, of the agonists tested, only ADP was able to stimulate thymidine uptake. Pretreatment with pertussis toxin abolished the PGE(2) response but had less of an effect on thrombin. PGE(2)- and thrombin-induced ERK1/2 activation was mimicked by 4-beta-phorbol-12-myristate-13-acetate and ionomycin and blocked by mitogen-activated protein kinase kinase inhibitor 1,4 diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene but displayed differential sensitivity to protein kinase C inhibitor bisindolylmaleimide I and Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Analogs of cAMP or agents that stimulate cAMP production were either weak or ineffective activators. Further studies indicate that the effect of thrombin was blocked by the phosphoinositide 3-kinase inhibitor wortmannin but not by agents inhibiting tyrosine kinase activity. On the contrary, herbimycin, but not wortmannin, attenuated the effect of PGE(2). Collectively, these results indicate that ERK1/2 are selectively activated by G protein-coupled receptors and not functionally associated with proliferation in HEL cells. ERK1/2 activation in response to PGE(2) and thrombin is mediated by distinctive types of G proteins and is differentially regulated by multiple pathways, including calcium mobilization, protein kinase C, phosphoinositide 3-kinase, and tyrosine kinases.

Calcium↗
PubMed2002

Selected contribution: Differential expression of stress-related genes with aging and hyperthermia.

Aging is associated with a reduced capacity to cope with physiological stress. To study the molecular mechanisms associated with the decline in stress tolerance that accompanies aging, differences in gene expression between young and old Fischer 344 rats under euthermic control conditions or in response to hyperthermic challenge were evaluated using a cDNA array containing 207 stress-related genes. In the nonstressed control condition, aging resulted in selective upregulation of stress protein genes and transcripts involved in cell growth, death, and signaling, along with a downregulation of genes involved in antioxidant defenses and drug metabolism. Heat stress resulted in a broad induction of genes in the antioxidant and drug metabolism categories and transcripts involved in DNA, RNA, and protein synthesis for both age groups. Old animals had a robust upregulation of genes involved in cell growth, death, and signaling after heat challenge, along with a blunted expression of stress-response genes. In contrast, young animals had a strong induction of stress-response genes after hyperthermic challenge. Changes in expression of selected genes were confirmed by RT-PCR analysis. These findings suggest that aging results in altered gene expression in response to heat stress that is indicative of decreased stress protein transcription and increased expression of oxidative stress-related genes. Thus our findings support the postulate that transcriptional changes in response to a physiological challenge such as hyperthermia contribute to the loss of stress tolerance in older organisms.

Aging↗
PubMed2003

Functional differentiation and selective inactivation of multiple Saccharomyces cerevisiae genes involved in very-long-chain fatty acid synthesis.

While de novo fatty acid synthesis uses acetyl-CoA, fatty acid elongation uses longer-chain acyl-CoAs as primers. Several mutations that interfere with fatty acid elongation in yeast have already been described, suggesting that there may be different elongases for medium- and long-chain acyl-CoA primers. In the present study, an experimental approach is described that allows differential characterization of the various yeast elongases in vitro. Based on their characteristic primer specificities and product patterns, at least three different yeast elongases are defined. Elongase I extends C12-C16 fatty acyl-CoAs to C16-C18 fatty acids. Elongase II elongates palmitoyl-CoA and stearoyl-CoA up to C22 fatty acids, and elongase III synthesizes 20-26-carbon fatty acids from C18-CoA primers. Elongases I, II and III are specifically inactivated in, respectively, elo1, elo2 and elo3 mutants. Elongases II and III share the same 3-ketoacyl reductase, which is encoded by the YBR159w gene. Inactivation of YBR159w inhibits in vitro fatty acid elongation after the first condensation reaction. Although in vitro elongase activity is absent, the mutant nevertheless contains 10-30% of normal VLCFA levels. On the basis of this finding, an additional elongating activity is inferred to be present in vivo. ybr159Delta cells show synthetic lethality in the presence of cerulenin, which inactivates fatty acid synthase. An involvement of FAS in VLCFA synthesis may account for these findings, but remains to be demonstrated directly. Alternatively, a vital role for C18 and C20 hydroxyacids, which are dramatically overproduced in ybr159Delta cells, may be postulated.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed1999

Analysis of the C5a anaphylatoxin core domain using a C5a phage library selected on differentiated U937 cells.

The human anaphylatoxin C5a is a 74-amino acid comprising polypeptide with a plethora of biological functions. Site directed mutagenesis studies suggest that several residues within the core and the C-terminus mediate the interaction with the C5a receptor. However, the contribution of particular core residues to receptor binding remained to be clarified. By means of the phage display technique, the loop between positions 35-40 was randomly mutated and the resulting C5a[35-40] fusion phage library affinity selected on C5a receptor expressing U937 cells. After five rounds of affinity enrichment, residues Arg37 and Arg40 were preferably selected. Enrichment was as high as 100% for Arg37 and 79% for Arg40. No significant enrichment of consensus residues could be obtained for positions 35, 36, 38 and 39. The core mutant C5a[A35E36R37A38S39R40], in which only Arg37/40 and Ala38 are of the native C5a sequence, was as potent as native C5a in both receptor binding and enzyme release examined on U937 cells. In contrast, replacement of Arg40 as in the mutant C5a[Q35E36R37I38L39N40] resulted in a 10-fold decrease in both binding and functional activities. Thus, selected out of a multiplicity of possibilities by the natural binding partner, Arg37 as well as Arg40 appear to be anchor residues in binding to the C5a receptor.

Antigens, CD↗