The amino acid sequence of major outer membrane protein II* (ompA protein) from Escherichia coli K-12 has been determined. The transmembrane polypeptide consists of 325 residues, resulting in a molecular weight of 35,159. The transmembrane part of the protein is located between residues 1 and 177. In Mis part of te rotein a predominantly lipophilic 27-residue segment exists that perhaps spans the membrane in a mostly a-helical conformation, or a 19-residue stretch of this segment might traverse the membrane linearly. Inside the outer membrane a sequence -Ala-Pro-AlaPro-Ala-Pro-Ala-Pro-exists that, analogous to the -Cys-Pro-ProCys-Pro-sequence in the hinge region of immunoglobulin, could assume the conformation of a polyproline helix. Computer analysis did not reveal a clear overall pattern of internal homology in the protein; besides the -Ala-Pro-repeat, only one local area (two adjacent dodeca eptide segments) shows some repetitiveness. The same analysis did not produce evidence for internal homology in the previously determined sequence of outer membrane protein I (porin) nor was any marked resemblance detected between transmembrane proteins I and II*.

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT 1-R͞cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT 2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD 4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT 2-R-and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at Ϸ60 min, were unaffected by a cysLT 1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD 4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT 2-R activation results in a proinflammatory EC phenotype. Because LTD 4 and thrombin are likely to be formed concomitantly in vivo, cysLT 2-R and PAR-1 may cooperate to augment vascular injury.cysteinyl leukotriene 2 receptor gene signature ͉ protease-activated receptor 1 gene signature ͉ vascular inflammation L eukotrienes (LTs), i.e., LTB 4 and the cysteinyl LTs (cysLT) LTC 4 , LTD 4 , and LTE 4 constitute a group of lipid mediators derived from the 5-lipoxygenase (5-LO) pathway (1, 2). LTs are either produced by leukocytes at sites of inflammation or formed through transcellular metabolism after uptake and metabolism of leukocyte-derived LTA 4 by downstream enzymes of the 5-LO pathway (LTA 4 hydrolase and LTC 4 synthase) in cells that normally do not express 5-LO, such as endothelial cells (ECs) (3, 4). LTs act through G protein-coupled surface receptors (GPCRs), i.e., the LTB 4 receptors and the cysLT receptors (LT-Rs) (cysLT 1 -R and cysLT 2 -R) (5-10). LT-Rs are expressed on multiple target cells, including leukocytes, smooth muscle cells, and ECs (1). Recent studies implicate the 5-LO pathway in cardiovascular disease (11)(12)(13)(14)(15)(16)(17).Considerable information is available on cysLT 1 -R, whereas little is known about cysLT 2 -R. We have used human umibilical vein (HUV)ECs as a model of vascular cells to study cysLT 2 -R activation by demonstrating that cysLTs exclusively signal through cysLT 2 -R in this cell type (18): In fact, HUVECs are the first primary cell type that s...

The amino acid sequence of the pore-forming outer membrane protein I (porin) from Escherichia coli B/r has been determined. The polypeptide contains 340 amino acid residues resulting in a molecular weight of 37,205. The transmembrane polypeptide has no stretches of nonpolar residues, uninterrupted by charged side chains, longer than 11 amino acid residues. Regarding polarity, the chain can be subdivided into three regions: a distinctly hydrophilic region between residues 1 and 82 (51.2% polarity), a fairly nonpolar region between residues 83 and 194 (33.9% polarity), and a more hydrophilic region up to the COOH terminus (48% polarity). These results are interpreted as evidence against a simple transmembrane structure in which the membrane is spanned by a single contiguous sequence of hydrophobic amino acids, as has been proposed, or example, for glycophorin. The cell envelope of Gram-negative bacteria possesses, in addition to the plasma membrane, an outer membrane which has perhaps more correctly also been called a porous skeletal organ (see ref. 1 for recent review) (2). This porosity is provided by proteins, the porins (e.g., refs. 3-5), which form hydrophilic channels allowing the diffusion of various low molecular weight solutes. In Escherichia coli B/r, protein 1 (6) [closely related to Rosenbusch's matrix protein from E. coli BE, (7)] is the porin responsible for the existence of these channels, which have a diameter of about 0.9 nm (8, 9).Aside from the fact that information is sparse regarding structure-function relationships of integral membrane proteins in general, the porins pose a number of interesting questions in addition to those connected with their physiological functions. In E. coli K-12 a whole family of such proteins of similar size and properties exists, and it seems that, under usual laboratory conditions, several of the corresponding structural genes are silent or nearly so (10-13). These genes are not clustered on the E. coli chromosome, and if they have arisen by duplications they might allow some insight into the evolution of this chromosome (11,(13)(14)(15)(16)(17) RESULTS AND DISCUSSION The amino acid sequence proposed for protein I (the porin from E. coli B/r) is shown in Fig. 1. The protein contains 340 amino acid residues and from this composition (Table 1) a molecular weight of 37,205 is calculated. The composition derived from the sequence and that calculated from amino acid analyses of acid hydrolyzates of the whole protein agree well (Table 1), and the molecular weight calculated from the sequence is in excellent agreement with earlier estimates (7). Protein I is the largest integral membrane polypeptide that has so far been sequenced.The primary structure of protein I was derived by analyses of peptides obtained by cleavage with cyanogen bromide (CNBr), trypsin, thermolysin, and the Staphylococcus aureus protease specific for glutamic acid residues (26). The strategy was first to determine the alignment of the four CNBr fragments (27) [CNBrl (38 residues), CNBr2 (76 ...

The amino acid sequence of the pore-forming outer membrane protein I (porin) from Escherichia coli B/r has been determined. The polypeptide contains 340 amino acid residues resulting in a molecular weight of 37,205. The trans-membrane polypeptide has no stretches of nonpolar residues, uninterrupted by charged side chains, longer than 11 amino acid residues. Regarding polarity, the chain can be subdivided into three regions: a distinctly hydrophilic region between residues 1 and 82 (51.2% polarity), a fairly nonpolar region between residues 83 and 194 (33.9% polarity), and a more hydrophilic region up to the COOH terminus (48% polarity). These results are interpreted as evidence against a simple transmembrane structure in which the membrane is spanned by a single contiguous sequence of hydrophobic amino acids, as has been proposed, or example, for glycophorin. The cell envelope of Gram-negative bacteria possesses, in addition to the plasma membrane, an outer membrane which has perhaps more correctly also been called a porous skeletal organ (see ref. 1 for recent review) (2). This porosity is provided by proteins, the porins (e.g., refs. 3-5), which form hydrophilic channels allowing the diffusion of various low molecular weight solutes. In Escherichia coli B/r, protein 1 (6) [closely related to Rosenbusch's matrix protein from E. coli BE, (7)] is the porin responsible for the existence of these channels, which have a diameter of about 0.9 nm (8, 9). Aside from the fact that information is sparse regarding structure-function relationships of integral membrane proteins in general, the porins pose a number of interesting questions in addition to those connected with their physiological functions. In E. coli K-12 a whole family of such proteins of similar size and properties exists, and it seems that, under usual laboratory conditions, several of the corresponding structural genes are silent or nearly so (10-13). These genes are not clustered on the E. coli chromosome, and if they have arisen by duplications they might allow some insight into the evolution of this chromosome (11, 13-17). Furthermore, several of these proteins can serve as at least parts of phage receptors (18-21) and they are required for an apparent uptake of protein into the cell: mutants lacking certain such polypeptides are highly tolerant to several colicins (e.g., refs. 22 and 23). It is not known what constitutes a phage receptor area on such proteins and what is their function in colicin sensitivity. Finding answers to all these and related questions would be helped by a knowledge of the amino acid sequence of such a protein. It is also likely that the gene for the protein under study will soon become available by DNA cloning. The determination of the DNA sequence, of much interest because of the unknown control region(s), should of course also be much aided by knowledge of the primary structure of the protein. We have determined the sequence of protein I from E. coli B/r and here present its primary structure. [Strains of E. coli B/r can dif...

A number of T-even-like bacteriophages use the outer membrane protein OmpA of Escherichia coli as a receptor. We had previously analyzed a series of ompA mutants which are resistant to such phages and which still produce the OmpA protein (R.