IRAK: a kinase associated with the interleukin-1 receptor.

The pleiotropic biological activities of interleukin-1 (IL-1) are mediated by its type I receptor (IL-1RI). When the ligand binds, IL-1RI initiates a signaling cascade that results in the activation of the transcription regulator nuclear factor kappa B (NF-kappa B). A protein kinase designated IRAK (IL-1 receptor-associated kinase) was purified, and its complementary DNA was molecularly cloned. When human embryonic kidney cells (cell line 293) over-expressing IL-1RI or HeLa cells were exposed to IL-1, IRAK rapidly associated with the IL-1RI complex and was phosphorylated. The primary amino acid sequence of IRAK shares similarity with that of Pelle, a protein kinase that is essential for the activation of a NF-kappa B homolog in Drosophila.

An interleukin-4-induced transcription factor: IL-4 Stat.

Interleukin-4 (IL-4) is an immunomodulatory cytokine secreted by activated T lymphocytes, basophils, and mast cells. It plays an important role in modulating the balance of T helper (Th) cell subsets, favoring expansion of the Th2 lineage relative to Th1. Imbalance of these T lymphocyte subsets has been implicated in immunological diseases including allergy, inflammation, and autoimmune disease. IL-4 may mediate its biological effects, at least in part, by activating a tyrosine-phosphorylated DNA binding protein. This protein has now been purified and its encoding gene cloned. Examination of the primary amino acid sequence of this protein indicates that it is a member of the signal transducers and activators of transcription (Stat) family of DNA binding proteins, hereby designated IL-4 Stat. Study of the inhibitory activities of phosphotyrosine-containing peptides derived from the intracellular domain of the IL-4 receptor provided evidence for direct coupling of receptor and transcription factor during the IL-4 Stat activation cycle. Such observations indicate that IL-4 Stat has the same functional domain for both receptor coupling and dimerization.

Identification of heregulin, a specific activator of p185erbB2.

The proto-oncogene designated erbB2 or HER2 encodes a 185-kilodalton transmembrane tyrosine kinase (p185erbB2), whose overexpression has been correlated with a poor prognosis in several human malignancies. A 45-kilodalton protein heregulin-alpha (HRG-alpha) that specifically induced phosphorylation of p185erbB2 was purified from the conditioned medium of a human breast tumor cell line. Several complementary DNA clones encoding related HRGs were identified, all of which are similar to proteins in the epidermal growth factor family. Scatchard analysis of the binding of recombinant HRG to a breast tumor cell line expressing p185erbB2 showed a single high affinity binding site [dissociation constant (Kd) = 105 +/- 15 picomolar]. Heregulin transcripts were identified in several normal tissues and cancer cell lines. The HRGs may represent the natural ligands for p185erbB2.

Cloning of AL-1, a ligand for an Eph-related tyrosine kinase receptor involved in axon bundle formation.

REK7 is an Eph-related tyrosine kinase receptor expressed exclusively in the nervous system, predominantly in hippocampus and cortex. A soluble REK7-IgG fusion protein, produced to analyze the biological role of REK7, prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. Using REK7-IgG as an affinity reagent, we purified and cloned a novel REK7 ligand called AL-1, a GPI-linked protein homologous to other members of an emerging ligand family. Membrane attachment of AL-1 appears necessary for receptor activation, since REK7 on cortical neurons is efficiently activated by transfected cells expressing GPI-linked AL-1, but not by soluble AL-1. Consistent with this, soluble AL-1 blocks axon bundling. Our findings, together with the observation that both molecules are expressed in the brain, suggest a role in the formation of neuronal pathways, a crucial feature of nervous system development and regeneration.

Cloning and expression of the growth hormone-dependent insulin-like growth factor-binding protein.

N-terminal as well as internal amino acid sequence data were obtained from the GH dependent, insulin-like growth factor (IGF) binding protein, BP-53, purified from human plasma. Based on these sequence data, full-length cDNA clones of BP-53 have been isolated, and the complete deduced sequence of BP-53 determined. This sequence contains a 27 amino acid putative signal sequence followed by a mature protein of 264 amino acids containing 18 cysteine residues clustered near the N- and C-terminus. The deduced protein sequence of BP-53 has 33% amino acid identity including conservation of all 18 cysteine residues with the recently cloned BP-28, a smaller human IGF-binding protein identified in amniotic fluid and also secreted by the cell line HEP G2. Expression of the cloned BP-53 cDNA in mammalian tissue culture cells results in secretion of the protein into the culture medium. This expressed protein is identical to plasma-derived BP-53 in its immunoreactivity, high affinity binding of IGF-I and IGF-II, and mobility on sodium dodecyl sulfate gel electrophoresis.

In vitro aging of calmodulin generates isoaspartate at multiple Asn-Gly and Asp-Gly sites in calcium-binding domains II, III, and IV.

We have determined the major sites responsible for isoaspartate formation during in vitro aging of bovine brain calmodulin under mild conditions. Protein L-isoaspartyl methyltransferase (EC 2.1.1.77) was used to quantify isoaspartate by the transfer of methyl-3H from S-adenosyl-L-[methyl-3H]methionine to the isoaspartyl (alpha-carboxyl) side chain. More than 1.2 mol of methyl-acceptor sites per mol of calmodulin accumulated during a 2-week incubation without calcium at pH 7.4, 37 degrees C. Analysis of proteolytic peptides of aged calmodulin revealed that > 95% of the methylation capacity is restricted to residues in the four calcium-binding domains, which are predicted to be highly flexible in the absence of calcium. We estimate that domains III, IV, and II accumulated 0.72, 0.60, and 0.13 mol of isoaspartate per mol of calmodulin, respectively. The Asn-97-Gly-98 sequence (domain III) is the greatest contributor to isoaspartate formation. Other major sites of isoaspartate formation are Asp-131-Gly-132 and Asp-133-Gly-134 in domain IV, and Asn-60-Gly-61 in domain II. Significant isoaspartate formation was also localized to Asp-20, Asp-22, and/or Asp-24 in domain I, to Asp-56 and/or Asp-58 in domain II, and to Asp-93 and/or Asp-95 in domain III. All of these residues are calcium ligands in the highly conserved EF-hand calcium-binding motif. Thus, other EF-hand proteins may also be subject to isoaspartate formation at these ligands. The results support the idea that isoaspartate formation in structured proteins is strongly influenced by both the C-flanking residue and by local flexibility.

Differential effects of insulin-like growth factor (IGF)-I and IGF-II on the expression of IGF binding proteins (IGFBPs) in a rat neuroblastoma cell line: isolation and characterization of two forms of IGFBP-4.

The isolation and hormonal regulation of two low molecular weight insulin-like growth factor binding proteins (IGFBPs) present in the conditioned medium (CM) of the rat neuroblastoma cell line B104 cells has been performed. IGFBPs were purified by ZnSO4 precipitation, insulin-like growth factor-I 1IGF-I) affinity chromatography, and reverse phase HPLC. Final isolation and N-terminal analysis was accomplished by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotting to polyvinylidene difluoride membranes, and sequencing of the bound proteins. Two IGFBPs, with apparent Mr of 28K and 24K were coisolated and sequenced. Both proteins had identical N-terminal sequences and appear to be two forms of IGFBP-4. Treatment of the IGFBPs with endoglycosidase-F caused a shift in the apparent Mr of the 28K IGFBP to 24K. However, there was no change in the apparent Mr of the 24K IGFBP. The data from this study suggest that the IGFBP-4 exists as both a glycosylated and nonglycosylated protein. Treatment of B104 cells with IGF-I increased the expression of both the 24K and 28K IGFBPs and also resulted in the appearance of IGFBP-3 and an unknown IGFBP at 29K. When added to subconfluent cells, IGF-I was also mitogenic in B104 cells. Similar to IGF-I, IGF-II treatment increased cell number and resulted in the appearance of IGFBP-3 and the 29K IGFBP. However, IGF-II treatment resulted in a significant decrease (approximately 50%) in the 24K IGFBP and also decreased the 28K IGFBP. This decrease in the expression of the 24K and 28K IGFBPs was dose-dependent and was blocked by addition of IGF-I to the cells. When an IGF-II receptor antibody was added to the cells it mimicked the effects of IGF-II on B104 cells, suggesting that the inhibitory effects of IGF-II are mediated through the type II IGF receptor. Although both IGF-I and IGF-II affected the amount of the 24K IGFBP in the CM, neither peptide affected the expression of the messenger RNA for the 24K IGFBP. In conclusion, we have isolated two IGFBPs from the CM of B104 cells. Both the 24K and 28K IGFBPs appear to be isoforms of the same protein, and sequence data suggest these proteins are two forms of IGFBP-4. IGF-I increases the expression of both of these IGFBPs, whereas IGF-II decreases their expression.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular cloning and characterization of human and murine DNase II.

We have cloned and sequenced novel cDNAs that encode human and murine DNase II, the acidic deoxyribonuclease. Sequence analysis predicts that huDNase II contains an N-terminal signal sequence and that mature DNase II has 344 residues with a calculated molecular mass of 38 032 Da. DNase II is a novel enzyme with no homologies to proteins of known function. Surprisingly, C. elegans appears to possess a family of DNase II homologs. Unlike DNase I-like enzymes that have tissue-specific expression patterns, huDNase II is ubiquituously expressed at low levels. When huDNase II is expressed in human 293 cells, we observe secretion of a novel 42-44 kDa glycoprotein; approximately 20-30% of recombinant human DNase II activity is secreted in this system. The secreted enzyme possesses DNA hydrolytic activity and shares biochemical properties with purified DNase II obtained from other species. We also show that the mechanism by which DNase II cuts DNA is similar to DNase I in that the enzyme produces nicks rather than double-strand cuts.

Micro quantitative Edman manual sequencing.

A manual Edman technique is described which allows sequential quantitative determination of from 3 to 10 amino terminal residues on quantities of peptides or proteins down to one nanomole. This is achieved by a fast, efficient method of obtaining the anilinothiazolinone or phenylthiohydantoin amino acid, and quantitating by either back hydrolysis and amino acid analysis or by a new, rapid, high resolution, quasi-isocratic, high-pressure liquid chromatographic procedure. The overall method has been extensively tested successfully on both peptides and proteins of known and unknown amino-terminal sequence and the results included here. In addition, a wide variety of applications relevant to primary structure analysis such as sequencing blocked polypeptides, use of denaturing agents as coupling buffers, reduction of protein or peptide losses on consecutive sequencing and peptide mixture analysis are all incorporated in the methodology outlined.

A carboxylesterase from the parasitic nematode Ascaris suum homologous to the intestinal-specific ges-1 esterase of Caenorhabditis elegans.

We have identified a carboxylesterase in A. suum that appears to be the homolog of the gut-specific C. elegans ges-1 enzyme. The A. suum esterase was purified and its N-terminal sequence found to be 50% identical to the C. elegans ges-1 protein. We have used isoelectric focusing analysis to demonstrate that, unlike the C. elegans ges-1 esterase, the A. suum enzyme is not restricted to the gut but is expressed in a wide range of tissues.

Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells.

A growth factor for vascular endothelial cells was identified in the media conditioned by bovine pituitary follicular cells and purified to homogeneity by a combination of ammonium sulfate precipitation, heparin-sepharose affinity chromatography and two reversed phase HPLC steps. The growth factor was a cationic, heat stable and relatively acid stable protein and had a molecular weight, as assessed by silver-stained SDS-PAGE gel, of approximately 45,000 under non reducing conditions and approximately 23,000 under reducing conditions. The purified growth factor had a maximal mitogenic effect on adrenal cortex-derived capillary endothelial cells at the concentration of 1-1.2 ng/ml (22-26 pM). Further characterization of the bioactivity of the growth factor reveals that it exerts mitogenic effects also on vascular endothelial cells isolated from several districts but not on adrenal cortex cells, lens epithelial cells, corneal endothelial cells, keratynocytes or BHK-21 fibroblasts, indicating that its target cells specificity is unlike that of any previously characterized growth factor. Microsequencing reveals a unique N-terminal amino acid sequence. On the basis of its apparent target cell selectivity, we propose to name this factor vascular endothelial growth factor (VEGF).

Matrix-assisted laser desorption/ionization time-of-flight mass analysis of peptides.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is one of the most useful techniques for determining the mass of biomolecules, with exceptional capabilities for mass analysis of peptides. Relative to other ionization techniques, it provides high sensitivity and excellent tolerance of salt and other common buffer components. Routine detection limits for peptides are in the subpicomole range. The ions commonly observed are the protonated molecules (M+H(+)), which makes data analysis relatively easy. This overview discusses instrument configuration and calibration, sample preparation, along with specific approaches for analyzing peptide mixtures, synthetic peptides, and chemical modifications of peptides.

Reversed-phase isolation of peptides.

In reversed-phase HPLC, peptides are separated on a hydrophobic stationary phase and eluted with a gradient of increasing organic solvent concentration. Protocols describing the separation of peptides in 5- to 500-pmol quantities via narrow-bore (2-mm-i.d.) or microbore (1-mm-i.d.) columns, as well as for the separation of peptides in quantities <5 pmol are provided in this unit. Capillary HPLC columns require a gradient flow rate of 3 to 5 omponents present in a small sample prior to automated sequencing is possible via the procedures for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and capillary electrophoresis.

Computer analysis of automated Edman degradation and amino acid analysis data.

Computer programs are described that allow facile analysis of data from a protein sequencer and amino acid analyzer. The sequencer program provides automated sequence interpretation while requiring minimal user interaction. The program serves as a powerful aid in deciphering mixture sequences and allows routine monitoring of sequencer performance. The computer program for amino acid analysis data provides the following calculations: mole percent, protein concentration and residues per mole with comparison between theoretical and calculated values. A plot of molecular weight versus deviation from integer values is calculated providing a measure of peptide or protein purity.

Photoaffinity labeling by 4-thiodideoxyuridine triphosphate of the HIV-1 reverse transcriptase active site during synthesis. Sequence of the unique labeled hexapeptide.

The active site of HIV-1 reverse transcriptase (HIV-1 RT) was investigated by photoaffinity labeling based on catalytic competence. A stable ternary elongation complex was assembled containing enzyme, DNA template (RT20), DNA primer molecule (P12), and the necessary dNTPs (one of which was alpha-32P-labeled) needed for primer elongation. The photoaffinity probe 4-thiodideoxyuridine triphosphate was incorporated uniquely at the 3' terminus of the 32P-labeled DNA product. Upon photolysis, the p66 subunit of a HIV-1 RT heterodimer (p66/p51) was uniquely cross-linked to the DNA product and subsequently digested by either trypsin or endoproteinase Lys-C. The labeled HIV-1 RT peptide was separated, purified, and finally subjected to Edman microsequencing. A unique radioactive hexapeptide (V276RQLCK281) was identified and sequenced. Our photoaffinity labeling results were positioned on the HIV-1 RT. DNA.Fab complex x-ray crystallography structure and compared with the suggested aspartic triad active site.

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry.

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.

Analysis of protein digests by capillary high-performance liquid chromatography and on-line fast atom bombardment mass spectrometry.

An HPLC system incorporating a packed capillary C18 column has been utilized for high sensitivity peptide mapping and preparative collection for protein sequencing. This system combined with a Frit-FAB mass spectrometer interface also provides the ability to obtain molecular ions for peptides of enzymatically digested proteins in the time it takes to obtain an HPLC chromatogram. The low flow rates permit introduction of the entire column effluent into the mass spectrometer. Detection limits of 0.5-5 pmol are routine. Proteolytic digests of recombinant human methionyl growth hormone and protein carboxyl methyltransferase have been used to demonstrate the HPLC and mass spectrometer performance.

Purification and sequence determination of canine relaxin.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.