Yeast pheromone alpha-factor is synthesized as a high molecular weight precursor

The sex pheromone alpha-factor of Saccharomyces cerevisiae, a tridecapeptide of approx. 1,700 molecular weight, was found to be synthesized in vivo as a high molecular weight precursor of Mr = 28,000. Inhibition of N-linked glycosylation by tunicamycin leads to three precursor species of lower molecular weight indicating three carbohydrate residues linked to the alpha-factor precursor molecule. A molecular weight of 18,000 was determined for the unglycosylated molecule.     

Incorporation of low molecular weight molecules into alpha(2)-macroglobulin by nucleophilic exchange

alpha(2)-Macroglobulin (alpha(2)M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (alpha(2)M( *)) can be employed to target antigens to antigen-presenting cells. Another potential use of alpha(2)M( *) is as a drug delivery system. In this study we demonstrate that guanosine triphosphate, labeled with Texas red (GTP-TR) formed complexes with alpha(2)M( *) following activation by proteolytic or non-proteolytic reactions. Optimal incorporation occurred with 20 microM GTP-TR, pH 8.0 for 5h at 50 degrees C. NaCl concentration (100 or 200 mM) had little effect on incorporation at this pH or temperature, but was significant at sub-optimum temperature and pH values. Maximum incorporation was 1.2 mol GTP-TR/mol alpha(2)M( *). PAGE showed that 70-90% of the GTP-TR is bound in a SDS/2-mercaptoethanol resistant manner. Guanosine, adenosine, and imidazole competed with GTP-TR to form complexes with alpha(2)M( *).     

Domain 5 of high molecular weight kininogen is antibacterial

Antimicrobial peptides are important effectors of the innate immune system. These peptides belong to a multifunctional group of molecules that apart from their antibacterial activities also interact with mammalian cells and glycosaminoglycans and control chemotaxis, apoptosis, and angiogenesis. Here we demonstrate a novel antimicrobial activity of the heparin-binding and cell-binding domain 5 of high molecular weight kininogen. Antimicrobial epitopes of domain 5 were characterized by analysis of overlapping peptides. A peptide, HKH20 (His(479)-His(498)), efficiently killed the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa and the Gram-positive Enterococcus faecalis. Fluorescence microscopy and electron microscopy demonstrated that HKH20 binds to and induces breaks in bacterial membranes. Furthermore, no discernible hemolysis or membrane-permeabilizing effects on eukaryotic cells were noted. Proteolytic degradation of high molecular weight kininogen by neutrophil-derived proteases as well as the metalloproteinase elastase from P. aeruginosa yielded fragments comprising HKH20 epitopes, indicating that kininogen-derived antibacterial peptides are released during proteolysis.     

Insertion of lipid domains into plasma membranes by fusion with erythrocytes

After prelabeling the plasma membrane with several lipid-specific fluorescent probes, erythrocytes with symmetric lipid bilayers were fused with culture cells using either poly(ethylene glycol) or Sendai virus as fusogen. Several nonspecific probes were transferred to, and became uniformly distributed within, the culture cell membrane upon fusion. In contrast, when merocyanine 540, which displays preferential binding to bilayers in which the lipids are loosely packed, was used to prelabel erythrocytes, fluorescence remained localized within a small confined area of the membrane, even 24 h after fusion. These results suggest that insertion of the lipids of the erythrocyte membrane into the plasma membrane of the culture cell can produce discrete domains which persist as such for long periods following fusion. Because the inserted proteins of the erythrocyte membrane similarly do not freely diffuse throughout the culture cell membrane, interactions between membrane proteins and lipids may be involved in this singular compartmentalization.     

Endothelial cell-derived high molecular weight von Willebrand factor is converted into the plasma multimer pattern by granulocyte proteases

We have previously found that the von Willebrand factor released by cultured human umbilical vein endothelial cells appeared as a single high molecular weight band in glyoxyl agarose electrophoresis. In the present studies we report that this high molecular weight endothelial cell-derived von Willebrand factor, when incubated with granulocyte lysates, was cleaved into a series of multimers indistinguishable from those seen in normal plasma (or type II von Willebrand disease). This von Willebrand factor-cleaving activity was released from granulocytes by calcium ionophore A23187 but was not detected in cytosolic fractions depleted of granular contents. It was inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. This von Willebrand factor-cleaving activity thus provides a possible mechanism for the generation of plasma von Willebrand factor multimers from the high molecular weight form of von Willebrand factor secreted by endothelial cells.     

Determination of molecular weight of membrane proteins by the use of low-angle laser light scattering combined with high-performance gel chromatography in the presence of a non-ionic surfactant

An assessment study was carried out to evaluate the performance of the low-angle laser light scattering technique combined with high-performance gel chromatography in the presence of a nonionic surfactant, octaethyleneglycol n-dodecyl ether, precision differential refractometry and ultraviolet photometry. It was found that the combined technique is highly promising as a method for the determination of the molecular weight of a membrane protein solubilized by the surfactant. For trial, molecular weights of the following membrane proteins of Escherichia coli, both solubilized in oligomeric forms, were measured; porin that forms the transmembrane diffusion pore in the outer membrane, and lambda-receptor protein that facilitates the diffusion of maltose-maltodextrins across the outer membrane. The result obtained indicates that both porin and lambda-receptor protein exist as trimers in the surfactant solution.     

Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions

A latent beta-TGF activity is spontaneously released into serum-free culture medium by chicken embryo fibroblasts. Anchorage-independent growth activity measured on NRK-49F indicator cells, of this latent beta-TGF can be revealed by four different treatments: acidification, alkalinisation, exposure to urea, and heating to 100 degrees C for 3 minutes. This lact activating treatment indicates that latent beta-TGF activation in vitro is non-enzymatic. Active beta-TGF exists in a low molecular weight form 16 Kd (apparent) in 1M acetic acid, which elutes on reverse phase (FPLC) between 33-35% acetonitrile. Under neutral conditions only a high molecular weight form excluded on Biogel P60 is observed. This form is poorly active on NRK-49F for anchorage independent growth but can be fully activated by prior acidification. Rechromatography of the latent beta-TGF-containing fractions under acidic conditions converts the high molecular weight form to an apparent 16 Kd active form. We suggest that the high molecular weight form may correspond to a complex of a beta-TGF associated with a carrier or binding protein.     

Increase of a high molecular weight protein in plasma membranes of BHK21 cells transformed by hamster sarcoma virus

Electrophoretic analysis of plasma membrane fractions from BHK21/13 cells non-transformed and transformed by the B34 strain of Hamster Sarcoma Virus showed a reproducible pattern of 18 main bands among which a band corresponding to a polypeptide of nominal molecular weight 177,000 daltons, is considerably increased in membranes of transformed cells. Labelling and solubilization studies suggest that it is an integral glycoprotein.     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Apoptotic effect of cleaved high molecular weight kininogen is regulated by extracellular matrix proteins

We previously reported that cleaved high molecular weight kininogen (HKa) and its domain 5 (D5) inhibit critical steps required for angiogenesis and in vivo neovascularization (Colman et al. 2000: Blood 95:543-550). We have further shown that D5 is able to induce apoptosis of endothelial cells, which may represent a critical part of the anti-angiogenic activity of HKa and D5 (Guo et al. 2001: Arterioscler Thromb Vasc Biol 21:1427-1433). In this study, we demonstrate that HKa- and D5-induced apoptosis is closely correlated with their anti-adhesive effect. An important new finding is that the apoptotic activity of HKa and D5 is highly regulated by their interactions with different extracellular matrix (ECM) proteins. HKa inhibited cell adhesion to vitronectin (Vn, 90%) and gelatin (Gel) (40%), but it had no apparent effect on cell adhesion to fibronectin (Fn). D5 showed a similar pattern on cell adhesion but was less potent than HKa. HKa induced apoptosis of endothelial cells grown on Vn and Gel but not cells grown on Fn which closely parallels with its anti-adhesive potency. Further results revealed that the anti-adhesive effect and the apoptotic effect of HKa are associated with its ability to inhibit phosphorylation of focal adhesion kinase (FAK) and paxillin, two important signal molecules required for cell adhesion and cell viability. We conclude that the anti-adhesive activity of HKa and D5 is responsible for their apoptotic effect and that Vn is likely an ECM component that mediates the effect of HKa and D5.     

Claudin-2 forms homodimers and is a component of a high molecular weight protein complex

Tight junctions are multiprotein complexes that form the fundamental physiologic and anatomic barrier between epithelial and endothelial cells, yet little information is available about their molecular organization. To begin to understand how the transmembrane proteins of the tight junction are organized into multiprotein complexes, we used blue native-PAGE (BN-PAGE) and cross-linking techniques to identify complexes extracted from MDCK II cells and mouse liver. In nonionic detergent extracts from MDCK II cells, the tight junction integral membrane protein claudin-2 was preferentially isolated as a homodimer, whereas claudin-4 was monomeric. Analysis of the interactions between chimeras of claudin-2 and -4 are consistent with the transmembrane domains of claudin-2 being responsible for dimerization, and mutational analysis followed by cross-linking indicated that the second transmembrane domains were arranged in close proximity in homodimers. BN-PAGE of mouse liver membrane identified a relatively discrete high molecular weight complex containing at least claudin-1, claudin-2, and occludin; the difference in the protein complex sizes between cultured cells and tissues may reflect differences in tight junction protein or lipid composition or post-translational modifications. Our results suggest that BN-PAGE may be a useful tool in understanding tight junction structure.     

Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli

Diffusely adhering Escherichia coli (DAEC) strains have been implicated in epidemiological studies as a cause of diarrhoea in children. However, the molecular interactions of these pathogens with target cells have remained largely obscure. We found that some DAEC strains contain homologues of the locus of enterocyte effacement (LEE) pathogenicity island and secrete EspA, EspB and EspD proteins necessary for the formation of the attaching and effacing (A/E) lesions. To characterize the function of the EspD protein further, we cloned and sequenced the espD genes of two DA-EPEC strains and compared their deduced amino-acid sequences with known EspD sequences. A pattern of two conserved transmembrane regions and one conserved coiled-coil region is predicted in EspD and also in the type III system secreted proteins YopB, PopB, IpaB and SipB of Yersinia, Pseudomonas, Shigella and Salmonella respectively. The EspD protein is inserted into a trypsin-sensitive location in the HeLa cell membrane at sites of bacterial contact, but is not translocated into the cytoplasm. Secretion of EspD increases upon contact with host cells. We propose that the membrane-located EspD protein is part of the translocation apparatus for Esp proteins into the target host cell performing functions similar to YopB in Yersinia.     

The mammalian primase is part of a high molecular weight DNA polymerase alpha polypeptide

The recently discovered eukaryotic primases have been found in tight association with certain DNA polymerase alpha forms. Here I present evidence that the high mol. wt. catalytic polypeptide (125,000) of an apparently homogeneous DNA polymerase alpha from freshly harvested calf thymus contains both polymerase and primase activity. This conclusion derives from the following three facts: (1) the two enzyme activities cannot be separated upon velocity sedimentation in 1.7 M urea, (2) both activities elute at a pI of 5.25 upon chromatofocussing and (3) after SDS-electrophoresis, renaturation of the enzymes in situ and measurement of DNA polymerase and primase activities in the gels, both enzymes have identical mobilities and coincide with the high mol. wt. catalytic subunit of DNA polymerase alpha.     

Insertion of anthrax protective antigen into liposomal membranes: effects of a receptor

Protective antigen (PA), the receptor-binding component of anthrax toxin, heptamerizes and inserts into the endosomal membrane at acidic pH, forming a pore that mediates translocation of the enzymic components of the toxin to the cytosol. When the heptameric pre-insertion form of PA (the prepore) is acidified in solution, it rapidly loses the ability to insert into membranes. To maximize insertion into model membranes, we examined two ways to bind the protein to large unilamellar vesicles (LUV). One involved attaching a His tag to the von Willebrand factor A domain of one of the PA receptors, ANTXR2, and using this protein as a bridge to bind PA to LUV containing a nickel-chelating lipid. The other involved using a His tag fused to the C terminus of PA to bind the protein directly to LUV containing the same lipid. Both ways enhanced pore formation at pH 5.0 strongly and about equally, as measured by the release of K+. Controls showed that pore formation in this system faithfully reproduced that in vivo. We also showed that binding unmodified ANTXR2 von Willebrand factor A to the prepore in solution enhanced its pore forming activity by slowing its inactivation at acidic pH. These findings indicate that an important role of PA receptors is to promote partitioning of PA into the bilayer by maintaining the prepore close to the target membrane and presumably in the optimal orientation as it undergoes the acidic pH-dependent conformational transition to the pore.     

Insertion of membrane proteins into discoidal membranes using a cell-free protein expression approach

We report a cell-free approach for expressing and inserting integral membrane proteins into water-soluble particles composed of discoidal apolipoprotein-lipid bilayers. Proteins are inserted into the particles, circumventing the need of extracting and reconstituting the product into membrane vesicles. Moreover, the planar nature of the membrane support makes the protein freely accessible from both sides of the lipid bilayer. Complexes are successfully purified by means of the apoplipoprotein component or by the carrier protein. The method significantly enhances the solubility of a variety of membrane proteins with different functional roles and topologies. Analytical assays for a subset of model membrane proteins indicate that proteins are correctly folded and active. The approach provides a platform amenable to high-throughput structural and functional characterization of a variety of traditionally intractable drug targets.     

Self-assembly of a high molecular weight basement membrane heparan sulfate proteoglycan into dimers and oligomers

A high molecular weight basement membrane heparan sulfate proteoglycan, isolated from murine Englebreth-Holm-Swarm tumor, is seen in platinum replicas as an elongated flexible core (Mr = 450,000) consisting of a series of tandem globular domains from which extend, at one end, two to three heparan sulfate chains (average Mr = 80,000 each). This macromolecule will self-assemble into dimers and lesser amounts of oligomers when incubated in neutral isotonic buffer. These molecular species can be separated by zonal velocity sedimentation and assembly is seen to be time- and concentration-dependent. In rotary-shadowed platinum replicas the binding region is found at or near the end of the core at the pole opposite the origin of the heparan sulfate chains. Dimers are double-length structures and oligomers are seen as stellate clusters: in both, the heparan sulfate chains appear peripherally oriented. While isolated cores self-assemble, isolated heparan sulfate chains do not bind intact proteoglycans. Furthermore, proteolytic removal of a non-heparan sulfate containing core moiety destroys the ability of the proteoglycan monomer to form larger species or bind intact proteoglycan, further supporting the binding topography determined morphologically. These negatively charged macromolecular complexes may be important contributors to basement membrane structure and function.     

Aggregation of liposomes by dextrans of high molecular weight

Sonicated dispersions (liposomes) of natural and synthetic phospholipids are aggregated reversibly by Dextrans 40, 110 and 500. The dextran concentration required for aggregation is dependent on chain length, lipid composition of the liposome and, for ionically-charged phospholipids, the ionic strength of the medium. The results indicate that adsorption of dextrans to the erythrocyte surface can occur by interaction with surface phospholipid substituents.     

Incorporation of high molecular weight RNA into large artificial lipid vesicles.

By utilizing an ether infusion technique with lecithin, cholesterol, and dicetylphosphate, giant liposomes have been produced with diameters ranging from 0.5 to 2.0 microns. These liposomes have been used to sequester 4S, 16S, and 23S $\text{E. coli}$ [³H]RNA and can be effectively separated from non-liposome incorporated RNA by ribonuclease treatment followed by Sepharose 4B gel filtration. The [³H]RNA within these liposomes can be extracted and appears to be undegraded.