Purification and complete sequence determination of the major plasma membrane substrate for cAMP-dependent protein kinase and protein kinase C in myocardium.

A protein of apparent Mr = 15,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is the major plasma membrane substrate for cAMP-dependent protein kinase (PK-A) and protein kinase C (PK-C) in several different tissues. In the work described here, we purified, cloned, and sequenced the canine cardiac sarcolemmal "15-kDa protein." The amino terminus of the purified protein was not blocked, allowing determination of 50 consecutive residues by standard Edman degradation. Overlapping proteolytic phosphopeptides yielded 22 additional residues at the carboxyl terminus. Dideoxy sequencing of the full-length cDNA confirmed that the 15-kDa protein contains 72 amino acids, plus a 20-residue signal sequence. The mature protein has a calculated Mr = 8409. There is one hydrophobic membrane-spanning segment composed of residues 18-37. The acidic amino-terminal end (residues 1-17) of the protein is oriented extracellularly, whereas the basic carboxyl-terminal end (residues 38-72) projects into the cytoplasm. The positively charged carboxyl terminus contains the phosphorylation sites for PK-A and PK-C. In the transmembrane region, the 15-kDa protein exhibits 52% amino acid identity with the "gamma" subunit of Na,K-ATPase. High stringency Northern blot analysis revealed that 15-kDa mRNA is present in heart, skeletal muscle, smooth muscle, and liver but absent from brain and kidney. We propose the name "phospholemman" for the 15-kDa protein, which denotes the protein's location within the plasma membrane and its characteristic multisite phosphorylation.     

Myristic acid is the NH2-terminal blocking group of the 43-kDa protein of Torpedo nicotinic post-synaptic membranes

The NH2-terminal blocking group of the 43-kDa peripheral membrane protein (43-kDa protein) of Torpedo post-synaptic membranes has been identified as myristic acid. To identify that blocking group pure 43-kDa protein was digested with trypsin and the blocked tryptic peptide was isolated by reverse phase HPLC. That peptide coeluted with and had the same amino acid composition as a synthetic peptide, myristoyl-Gly-Gln-Asp-Gln-Thr-Lys, the structure of the amino terminus predicted from the protein sequence deduced from a cDNA clone. The presence of myristate was confirmed by the precise molecular mass of the peptide, 886.5266, determined by fast atom bombardment mass spectroscopy.     

Protein and cDNA sequence of a glycine-rich, dimethylarginine-containing region located near the carboxyl-terminal end of nucleolin (C23 and 100 kDa)

By a combination of protein chemistry and recombinant DNA methods a glycine-rich region was found to be located near the carboxyl terminus of the nucleolar specific phosphoprotein, nucleolin, from Novikoff hepatoma (protein C23) and Chinese hamster ovary cells (100-kDa nucleolar protein). A sequence of 192 amino acid residues was derived from partial sequences of cyanogen bromide and N-bromosuccinimide fragments of protein C23 and deduced protein sequence from Chinese hamster ovary cell 100-kDa cDNA sequences. The 66 residues sequenced by protein methods were identical to the corresponding residues deduced by DNA sequencing. The multiple residues of NG,NG-dimethylarginine (DMA) contained in the nucleolin polypeptide were found to be limited to a segment of less than 10 kDa near the carboxyl-terminal end of the protein. This segment also contained internally repeated sequences (e.g. 7 copies of the sequence Gly-Gly-Arg-Gly-Gly were found) which were unrelated to sequences closer to the amino-terminal end. Most arginine residues in this region were surrounded by 2 or 3 glycine residues and were relatively close in sequence to phenylalanine residues.     

cDNA cloning and expression of a bovine phenol UDP-glucuronosyltransferase, BovUGT1A6

A full-length cDNA encoding a phenol UDP-glucuronosyltransferase was isolated by plaque hybridization, RT-PCR and 5'-RACE from a cDNA library prepared from the bovine liver. The deduced amino acid sequence (529 amino acid residues) has A signal sequence (23 amino acid residues) at the amino terminus and a transmembrane-anchoring domain (17 amino acid residues) at the carboxyl terminus. The encoded protein has a potential asparagine-linked glycosylation site (Asn291). The cloned cDNA was named bovUGT1A6 on the basis of the amino acid similarity. BovUGT1A6 cloned in the pAAH5 expression vector was transformed into Saccharomyces cerevisiea AH22 cells to obtain an active 54-kDa bovUGT1A6 enzyme. The expressed enzyme represented UDP-glucuronosyltransferase activities toward 1-naphthol and 4-methylumbelliferone, confirming that the isolated cDNA is an isoform of bovine phenol UDP-glucuronosyltransferase. Microsomal UDP-glucuronosyltransferase activity toward 1-naphthol in the bovine kidney cortex was found to be higher than that in the liver and other organs, and mRNA of bovUGT1A6 was more strongly detected in the kidney on Northern blotting analysis. These results suggest that the bovine kidney, which strongly expresses bovUGT1A6, is a significant organ for xenobiotics glucuronidation.     

The role of the polar, carboxyl-terminal domain of Escherichia coli leader peptidase in its translocation across the plasma membrane

Leader peptidase, an integral membrane protein of Escherichia coli, is made without a cleavable leader sequence. It has 323 amino acid residues and spans the plasma membrane with a small amino-terminal domain exposed to the cytoplasm and a large, carboxyl-terminal domain exposed to the periplasm. We have investigated which regions of leader peptidase are necessary for its assembly across the membrane. Deletions were made in the carboxyl-terminal domain of leader peptidase, removing residues 141-222, 142-323, or 222-323. Protease accessibility was used to determine whether the polar, carboxyl-terminal domains of these truncated leader peptidases were translocated across the membrane. The removal of either residues 222-323 (the extreme carboxyl terminus) or residues 141-222 does not prevent leader peptidase membrane assembly. However, leader peptidase lacking both regions, i.e. amino acid residues 142-323, cannot translocate the remaining portion of its carboxyl terminus across the membrane. Our data suggest that the polar, periplasmic domain of leader peptidase contains information which is needed for membrane assembly.     

Deletion analysis of the 51-kilodalton protein of the Bacillus sphaericus 2362 binary mosquitocidal toxin: construction of derivatives equivalent to the larva-processed toxin.

Bacillus sphaericus 2362 produces a binary toxin consisting of 51- and 42-kDa proteins, both of which are required for toxicity to mosquito larvae. Upon ingestion by larvae, these proteins are processed to 43 and 39 kDa, respectively. Using site-directed mutagenesis, we have obtained N- and C-terminal deletions of the 51-kDa protein and expressed them in B. subtilis by using the subtilisin promoter. Removal of 21 amino acids from the N terminus and 53 amino acids from the C terminus resulted in a protein with the same electrophoretic properties as the 43-kDa degradation product which accumulates in the guts of mosquito larvae. This protein was toxic only in the presence of the 42-kDa protein. A deletion of 32 amino acids at the N terminus combined with a 53-amino-acid deletion at the C terminus resulted in a protein which retained toxicity. Toxicity was lost upon a further deletion of amino acids at potential chymotrypsin sites (41 at the N terminus, 61 at the C terminus). Comparison of the processing of the 51- and the 42-kDa proteins indicated that in spite of their sequence similarity proteolysis occurred at different sites.     

Unique precursor structure of an extracellular protease, aqualysin I, with NH2- and COOH-terminal pro-sequences and its processing in Escherichia coli

Aqualysin I is a subtilisin-type serine protease which is secreted into the culture medium by Thermus aquaticus YT-1, an extremely thermophilic Gram-negative bacterium. The nucleotide sequence of the entire gene for aqualysin I was determined, and the deduced amino acid sequence suggests that aqualysin I is produced as a large precursor, consisting of at least three portions, an NH2-terminal pre-pro-sequence (127 amino acid residues), the protease (281 residues), and a COOH-terminal pro-sequence (105 residues). When the cloned gene was expressed in Escherichia coli cells, aqualysin I was not secreted. However, a precursor of aqualysin I lacking the NH2-terminal pre-pro-sequence (38-kDa protein) accumulated in the membrane fraction. On treatment of the membrane fraction at 65 degrees C, enzymatically active aqualysin I (28-kDa protein) was produced in the soluble fraction. When the active site Ser residue was replaced with Ala, cells expressing the mutant gene accumulated a 48-kDa protein in the outer membrane fraction. The 48-kDa protein lacked the NH2-terminal 14 amino acid residues of the precursor, and heat treatment did not cause any subsequent processing of this precursor. These results indicate that the NH2-terminal signal sequence is cleaved off by a signal peptidase of E. coli, and that the NH2- and COOH-terminal pro-sequences are removed through the proteolytic activity of aqualysin I itself, in that order. These findings indicate a unique four-domain structure for the aqualysin I precursor; the signal sequence, the NH2-terminal pro-sequence, mature aqualysin I, and the COOH-terminal pro-sequence, from the NH2 to the COOH terminus.     

Clusters in an intrinsically disordered protein create a protein-binding site: the TolB-binding region of colicin E9

The 61-kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins and kills them by hydrolyzing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies [Collins et al. (2002) J. Mol. Biol. 318, 787-904; MacDonald et al. (2004), J. Biomol. NMR 30, 81-96] have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is intrinsically disordered and contains clusters of interacting side chains. To further define the properties of this region of colicin E9, we have investigated the effects on the dynamical and TolB-binding properties of three mutations of colicin E9 that inactivate it as a toxin. The mutations were contained in a fusion protein consisting of residues 1-61 of colicin E9 connected to the N terminus of the E9 DNase by an eight-residue linking sequence. The NMR data reveals that the mutations cause major alterations to the properties of some of the clusters, consistent with some form of association between them and other more distant parts of the amino acid sequence, particularly toward the N terminus of the protein. However, (15)N T(2) measurements indicates that residues 5-13 of the fusion protein bound to the 43-kDa TolB remain as flexible as they are in the free protein. The NMR data point to considerable dynamic ordering within the intrinsically disordered translocation domain of the colicin that is important for creating the TolB-binding site. Furthermore, amino acid sequence considerations suggest that the clusters of amino acids occur because of the size and polarities of the side chains forming them influenced by the propensities of the residues within the clusters and those immediately surrounding them in sequence space to form beta turns.     

Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope.

The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.     

Isolation, properties, and the complete amino acid sequence of a second form of 60-kDa glycoprotein esterase. Orientation of the 60-kDa proteins in the microsomal membrane

A second form (form 2) of glycosylated esterase was isolated from liver microsomal membranes and characterized. The subunit molecular weight of form 2 is identical to that of the 60-kDa protein previously reported (Ozols, J. (1987) J. Biol. Chem. 262, 15316-15321). The NH2 terminus of the form 2 enzyme is blocked. Digestion of form 2 with pyroglutamyl aminopeptidase, followed by electroblotting and sequence analysis of the blotted protein, indicated that a pyroglutamyl residue was located at the NH2 terminus. Sequence analysis of the deblocked protein as well as characterization of the peptides obtained from enzymatic and chemical cleavages of the intact protein led to the elucidation of its complete amino acid sequence. The protein is a single polypeptide consisting of 532 residues. Carbohydrate is attached at asparaginyl residue 249. The sequence of form 2 esterase is 50% identical to the sequence of form 1 enzyme. The amino acid sequence of the first 26 residues of form 1 enzyme from human liver microsomes shows that 23 residues are identical to that of rabbit form 1, but only 8 residues that are identical to form 2. Treatment of the forms 1 and 2 isozymes with N-glycosidase F or endo-N-acetylglucosaminidase H resulted in a decrease of their subunit molecular weights, indicating that the carbohydrate attached is of the high mannose type. To determine whether the 60-kDa proteins are located on the cytoplasmic or luminal side of the endoplasmic membrane, microsomes were treated with proteolytic enzymes and the two 60-kDa isozymes were isolated and characterized. Sequence analysis of both proteins indicated that their NH2 termini were unaffected by proteolysis. Form 1 isozyme isolated from trypsin-treated microsomes, however, lacked the COOH-terminal heptapeptide (residues 533-539). These results, in addition to the finding of an N-linked carbohydrate, suggest that the two 60-kDa proteins are oriented on the luminal side of the endoplasmic membrane.     

Expression of the Chlamydia trachomatis major outer membrane protein-encoding gene in Escherichia coli: role of the 3' end in mRNA stability

The major outer membrane protein (MOMP)-encoding gene (omp1) of Chlamydia trachomatis has been cloned into Escherichia coli and partially sequenced. This recombinant gene expresses a full-length 40-kDa product, which is recognized by a monoclonal antibody directed against the species-specific epitope of MOMP. The recombinant omp1 is expressed in either insertion orientation, indicating that it utilizes its own promoter system. The endogenous omp1 promoter possesses a relatively low activity despite the high level of MOMP expression. Deletion of a 520-bp fragment at the 3' end encoding 39 amino acids (aa) at the C terminus and the remainder of the noncoding region leads to a significant decrease in mRNA stability and loss of protein synthesis. When the MOMP-encoding plasmid was introduced into E. coli minicells, it expressed 40- and 43-kDa proteins; however, inhibition of post-translational processing by ethanol revealed only a 43-kDa protein. These data indicate that the unprocessed omp1 gene product contains a 22-aa leader sequence which is cleaved during translocation to the outer membrane, to yield a processed 40-kDa protein. The recombinant MOMP was localized to the outer membrane E. coli fraction, comparable to the location of the native C. trachomatis protein.     

Structure of the cel-3 gene from Fibrobacter succinogenes S85 and characteristics of the encoded gene product, endoglucanase 3.

The cel-3 gene cloned from Fibrobacter succinogenes into Escherichia coli coded for the enzyme EG3, which exhibited both endoglucanase and cellobiosidase activities. The gene had an open reading frame of 1,974 base pairs, coding for a protein of 73.4 kilodaltons (kDa). However, the enzyme purified from the osmotic shock fluid of E. coli was 43 kDa. The amino terminus of the 43-kDa protein matched amino acid residue 266 of the protein coded for by the open reading frame, indicating proteolysis in E. coli. In addition to the 43-kDa protein, Western immunoblotting revealed a 94-kDa membranous form of the enzyme in E. coli and a single protein of 118 kDa in F. succinogenes. Thus, the purified protein appears to be a proteolytic degradation product of a native protein which was 94 kDa in E. coli and 118 kDa in F. succinogenes. The discrepancy between the molecular weight expected on the basis of the DNA sequence and the in vivo form may be due to anomalous migration during electrophoresis, to glycosylation of the native enzyme, or to fatty acyl substitution at the N terminus. One of two putative signal peptide cleavage sites bore a strong resemblance to known lipoprotein leader sequences. The purified 43-kDa peptide exhibited a high Km (53 mg/ml) for carboxymethyl cellulose but a low Km (3 to 4 mg/ml) for lichenan and barley beta-glucan. The enzyme hydrolyzed amorphous cellulose, and cellobiose and cellotriose were the major products of hydrolysis. Cellotriose, but not cellobiose, was cleaved by the enzyme. EG3 exhibited significant amino acid sequence homology with endoglucanase CelC from Clostridium thermocellum, and as with both CelA and CelC of C. thermocellum, it had a putative active site which could be aligned with the active site of hen egg white lysozyme at the highly conserved amino acid residues Asn-44 and Asp-52.     

Amino acid sequence of bovine white matter proteolipid

The sequence of the bovine white matter proteolipid has been studied by a combination of proteolytic digestion and chemical cleavage at tryptophan residues. Alignment of peptides obtained by digestion with trypsin, chymotrypsin, clostripain, and Staphylococcus aureus protease gave the sequence of 52 residues at the amino terminus, 96 residues at the carboxyl terminus, and several additional segments. Peptides obtained by treatment of the protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine confirmed the alignment and extended the sequence. This information, combined with that of other investigators, permits us to propose the primary structure for the entire protein. On the basis of the sequence determination, the molecular weight of the proteolipid protein is 29,869.     

Deletion of approximately 10 kDa from the carboxyl terminus of a soluble approximately 48-kDa insulin receptor protein-tyrosine kinase results in slower rates of diphosphorylation of a series of dodecapeptide substrates. An assessment by 1H NMR.

Autophosphorylation of a soluble approximately 48-kDa derivative of the insulin receptor protein-tyrosine kinase is accompanied by an increase in its specific activity towards exogenous substrates. In the present study, we have utilized 1H NMR to compare the order and rate of mono- and diphosphorylation of multiple tyrosine residues in a series of synthetic dodecapeptide substrates (based on the receptor sequence, which includes major sites of autophosphorylation (RRDIYETDYYRK), with substitution(s) at positions 6 and/or 7 based on residue size and/or charge) by the approximately 48-kDa enzyme and by a approximately 38-kDa enzyme generated by tryptic deletion of approximately 10 kDa from the carboxyl terminus of the approximately 48-kDa protein. Both enzymes exhibit a marked order and progression of phosphorylation of peptide tyrosine residues; for each peptide, phosphorylation initiates and proceeds to completion first on tyrosine 9, followed by phosphorylation on tyrosine 10. Although removal of the carboxyl terminus does not affect the rate of monophosphorylation of these peptides on tyrosine 9, the smaller enzyme exhibits a slower rate of diphosphorylation (at tyrosine 10), as compared with the approximately 48-kDa enzyme.