Glycation of MP26 and MP22 in bovine lens membranes.

Alkali treated membranes were isolated from mature bovine lenses and incubated with different sugars for 3 weeks to study the effect of glycation on the lens intrinsic membrane proteins, MP26 and MP22. The obtained results show that a) [1-14C] ascorbic acid (ASA) was able to glycate the intrinsic membrane proteins as rapidly as soluble lens proteins; b) on 15% acrylamide gels in SDS, glucose, fructose, galactose and ribose exhibited low activity for crosslinking membrane proteins; whereas ASA, dehydroascorbate (DHA), diketogulonate (DKG), xylosone and threose, all showed not only the formation of protein multimers, but also highly crosslinked products, which did not enter the spacer gel; c) except glycated MP22, all of the crosslinks of MP26 or MP22, and also the glycated MP26, showed cross reactivity with polyclonal MP26 antibody; d) the extent of crosslinking correlated with an equal loss of lysine and arginine contents by amino acid analysis.     

In vitro synthesis and membrane insertion of bovine MP26, an integral protein from lens fiber plasma membrane

Synthesis of MP26, the principal protein of lens fiber plasma membranes, was directed in the reticulocyte lysate system by poly A mRNA enriched from whole bovine lens RNA using oligo (dt)-cellulose chromatography. Synthesized MP26 was enriched by immune precipitation. The in vitro-synthesized MP26 had an electrophoretic mobility indistinguishable from that of the native molecule. MP26 showed a cotranslational requirement for dog pancreas microsomes in order for membrane association to occur. Microsome-associated in vitro- synthesized MP26 showed a sensitivity to digestion with chymotrypsin which was similar to the sensitivity of native MP26 in isolated lens fiber plasma membranes, indicating correct insertion of the MP26 into the microsome. Synthesis and membrane insertion of MP26 using N-formyl- [35S]methionyl tRNA as label demonstrated that no proteolytic processing or significant glycosylation accompanied membrane insertion. Chymotryptic cleavage of membrane-inserted, N-formyl-[35S]methionine- labeled MP26 resulted in loss of label, suggesting that the N-terminal of the in vitro-synthesized MP26 faces the cytoplasm.     

The permeability of reconstituted liposomes containing the purified lens fiber cell integral membrane proteins MP20, MP26 and MP70

Summary A number of lens fiber cell integral membrane proteins have been localized to junctional regions where they have been proposed to play a role in either mediating or controlling cell-to-cell communication. We have examined the effect of three lens fiber cell membrane proteins, MP20, MP26 and MP70, on the permeability properties of unilamellar phospholipid liposomes. This approach has been previously used to examine the channel-forming properties of MP26. Liposome permeability was determined by measuring the effect of Co²⁺ on the quenching of the fluorescence of N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidyl ethanolamine (NBD-PE)-containing liposomes as described previously by Scaglione and Rintoul (Invest. Ophthalmol. Vis. Sci. 30:961–966, 1989). The effect of all three proteins on liposome permeability was similar. Permeability was dependent on the protein/phospholipid ratio and was not significantly affected by agents known to modify gap junctional permeability in vivo. Glycophorin A, a non-channel-forming integral membrane protein derived from erythrocytes, was also shown to increase the permeability of unilamellar phospholipid liposomes. The ability of a non-channel membrane protein to increase Co²⁺ quenching of NBD-PE-containing liposomes (presumably in a nonspecific manner) indicates that reports describing the permeability of lens membrane protein-containing liposomes should be interpreted with caution in terms of their relationship to cell-to-cell communication.We would like to thank Dr. Rita Meyer for technical assistance with the freeze-fracture electron microscopy, Drs. Wolfgang Baumann and Barbara Malewicz for the purification of bovine lens lipids, and Dr. Gary Nelsestuen for the use of both the fluorescence and photon correlation spectrophotometers as well as for many helpful discussions. This research was supported by NIH grant EY 05684.     

Preparation, characterization, and localization of antisera against bovine MP26, an integral protein from lens fiber plasma membrane

Polyclonal antisera were prepared in rabbits using both native and chymotrypsin-digested bovine lens fiber plasma membranes. MP26, the principal protein of lens fiber plasma membranes, and CT20, a chymotryptic fragment of MP26, were isolated electrophoretically and used to purify anti-MP26 and anti-CT20 activity from the respective antisera by affinity chromatography. These affinity-purified antisera were characterized by immunoreplica. Immunofluorescence microscopy localized MP26 on sections of methacrylate-embedded lenses in the lens fiber plasma membranes, but not the lens epithelium. Immunocytochemistry of isolated native or chymotrypsin-digested lens fiber plasma membranes localized both the MP26 and the CT20 only in the nonjunctional plasma membranes, with no detectable activity in the lens fiber junctions themselves. Electron microscopy revealed a second set of pentalaminar profiles, thinner by 4 nm than the lens fiber junctions, which contained demonstrable anti-MP26 and anti-CT20 activity following immunocytochemistry. These results indicate either that MP26 is not a component of the lens fiber junctions, or that significant conformational changes accompany assembly of MP26 into lens fiber junctions, resulting in the masking of MP26 antigenic determinants.     

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.     

Structural organization of the lens fiber cell plasma membrane protein MP18

The 18,000-dalton bovine lens fiber cell intrinsic membrane protein MP18 was phosphorylated on a serine residue by both cAMP-dependent protein kinase and protein kinase C. In addition, this protein bound calmodulin and was recognized by a monoclonal antibody (2D10). These different regions were localized using enzymatic and chemical fragmentation of electrophoretically purified MP18 that had been phosphorylated with either cAMP-dependent protein kinase or protein kinase C. Partial digestion of 32P-labeled MP18 with protease V8 resulted in a Mr = 17,000 peptide that bound calmodulin, but neither contained 32P or was recognized by the monoclonal antibody 2D10. Furthermore, the 17-kDa peptide had the same N-terminal amino acid sequence as MP18. Thus, the monoclonal antibody 2D10 recognition site and the protein kinase phosphorylation site(s) are close together and confined to a small region in the C terminus of MP18. This conclusion was confirmed in experiments where MP18 was fragmented with trypsin, endoproteinase Lys-C, or CNBr. The location of the phosphorylation site was confirmed by sequencing the small 32P-labeled, C-terminal peptide that resulted from protease V8 digestion of 32P-labeled MP18. This peptide contained a consensus sequence for cAMP-dependent protein kinase.     

Major intrinsic polypeptide (MIP26K) from lens membrane: reconstitution into vesicles and inhibition of channel forming activity by peptide antiserum

Bovine and human lens membrane, when reconstituted into lipid vesicles containing oxidized cytochrome C, will mediate the transmembrane passage of ascorbate into the vesicles, where the reduction of cytochrome C is measured spectrophotometrically. This channel forming activity is specifically inhibited by antiserum made against a synthetic octapeptide near the C-terminus of MIP26K. Together, these studies describe a direct and more sensitive assay system for measurement of channel-forming activity of MIP26K, and suggest that the C-terminus of this molecule may be particularly important in the regulation of channel formation.     

Fatty acid acylation of lens fiber plasma membrane proteins. MP26 and alpha-crystallin are palmitoylated

We describe in this report the fatty acylation of some of the main polypeptides from the eye lens fibers. MP26, the major lens fiber plasma membrane protein, and probably MP22, its natural degradation product, are palmitoylated in a post-translational process. This is also the case for alpha-crystallin, a major cytoplasmic structural protein shown to interact directly with the plasma membrane. Furthermore, a 65 kDa non-identified polypeptide and a high molecular weight component are also modified in the same way.     

Stimulation and inhibition by magnesium ions of intrinsic protein phosphorylating systems in synaptosomal membrane fragments from rat brain

Synaptosomal membrane fragments from rat brain were incubated with [-³²P]ATP in the presence of cyclic AMP or Ca²⁺ plus calmodulin and a range of Mg²⁺ concentrations. Incorporation of³²P into membrane polypeptides was examined by electrophoresis and radioautography. Cyclic AMP-stimulated reactions were stimulated by low concentrations and inhibited to varying degrees by high concentrations of Mg²⁺ in the range 1–50 mM. In general the Ca²⁺ plus calmodulin-stimulated reactions were maximally active in the range 30–50 mM Mg²⁺, but the Ca²⁺ plus calmodulin dependent phosphorylation of Protein I was progressively inhibited by concentrations of Mg²⁺ above 5 mM. These results emphasize the importance of establishing optimum Mg²⁺ concentrations in the study of specific membrane protein phosphorylating systems.     

Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin.

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.     

Conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes

The conformational properties of the main intrinsic polypeptide (MIP26) isolated from lens plasma membranes were studied by using near- and far-ultraviolet circular dichroism. The far-ultraviolet spectrum of MIP26 solubilized with octyl beta-D-glucopyranoside indicates an alpha-helical content of approximately 50% and a beta-structure content of approximately 20%. A detergent-free membrane suspension of MIP26 produced a typically distorted far-ultraviolet spectrum which was caused by differential light scattering and absorption flattening. However, decreasing the size of the membrane fragments by sonication produced a far-ultraviolet spectrum free of distortion, and with a rotatory strength profile similar to that obtained for MIP26 solubilized with octyl beta-D-glucopyranoside. This implies similar secondary structure properties for the protein in both the suspension and the sugar detergent. The cleavage of MIP26 with Staphylococcus aureus protease, which results in removal of a 5-kilodalton peptide and which mimics the age-dependent posttranslational changes that take place in the lens, did not significantly affect the conformation of the core protein as judged by the near-ultraviolet circular dichroism spectra.     

Properties of channels reconstituted from the major intrinsic protein of lens fiber membranes

Detergent-solubilized plasma membrane protein of either adult bovine or calf lens and high-performance liquid chromatography-purified major intrinsic protein (MIP) of the lens were reconstituted into unilamellar vesicles and planar lipid bilayers. Freeze-fracture studies showed that the density of intramembrane particles in the vesicles was proportional to the protein/lipid ratio. At high ratios, these particles crystallized into tetragonal arrays as does MIP in lens fibers. Channels induced by either purified MIP or detergent-solubilized protein had essentially identical properties. The conductance of multichannel membranes was maximal near 0 mV and decreased to 0.49 +/- 0.08 of the maximum value at voltages greater than 80 mV. The dependence of the conductance on voltage was well fit by a two-state Boltzmann distribution. Voltage steps greater than 30 mV elicited an ohmic current step followed by a slow (seconds) biexponential decrease. The amplitudes and time constants depended on the magnitude but not the sign of the voltage. Steps from 100 mV to voltages less than 30 mV caused the channels to open exponentially with a millisecond time constant. Analysis of latency to first closure after a voltage step gave nearly the same time constants as multichannel kinetics. Single-channel conductance is proportional to salt concentration from 0.1 to 1.0 M in KCl. In 0.1M KCl, the channel had two preferred conductance states with amplitudes of 380 and 160 pS, as well as three additional substates. Multi- and single-channel data suggest that the channel has two kinetically important open states. The channel is slightly anion selective. The properties of the channel do not vary appreciably from pH 7.4 to 5.8 or from pCa 7 to 2. We propose that a channel with these properties could contribute to maintenance of lens transparency and fluid balance.     

Interaction of ATPase from submitochondrial fragments and a natural inhibitor protein during delta-mu-H+ generation on a membrane

An addition of the inhibitor protein (IF1) to submitochondrial particles (SMP) essentially free of endogenous IF1 (AS-SMP) results in a synchroneous inhibition of ATP hydrolysis and ATP-dependent reduction of NAD+ by succinate without any effect on the oxidative phosphorylation rate. The binding of IF1 to the membrane-bound ATPase leads to the loss of the inhibitor protein sensitivity to trypsin despite the delta mu H+ generation. The data obtained are consistent with a model according to which there exist the hydrolase and synthetase forms of F1 and contradict the generally accepted concepts on the delta mu H+-dependent dissociation of the F1-IF1 complex.     

Modular analysis of hipposin,a histone-derived antimicrobial peptide consisting of membrane translocating and membrane permeabilizing fragments

Antimicrobial peptides continue to garner attention as potential alternatives to conventional antibiotics. Hipposin is a histone-derived antimicrobial peptide (HDAP) previously isolated from Atlantic halibut. Though potent against bacteria, its antibacterial mechanism had not been characterized. The mechanism of this peptide is particularly interesting to consider since the full hipposin sequence contains the sequences of parasin and buforin II (BF2), two other known antimicrobial peptides that act via different antibacterial mechanisms. While parasin kills bacteria by inducing membrane permeabilization, buforin II enters cells without causing significant membrane disruption, harming bacteria through interactions with intracellular nucleic acids. In this study, we used a modular approach to characterize hipposin and determine the role of the parasin and buforin II fragments in the overall hipposin mechanism. Our results show that hipposin kills bacteria by inducing membrane permeabilization, and this membrane permeabilization is promoted by the presence of the N-terminal domain. Portions of hipposin lacking the N-terminal sequence do not cause membrane permeabilization and function more similarly to buforin II. We also determined that the C-terminal portion of hipposin, HipC, is a cell-penetrating peptide that readily enters bacterial cells but has no measurable antimicrobial activity. HipC is the first membrane active histone fragment identified that does not kill bacterial or eukaryotic cells. Together, these results characterize hipposin and provide a useful starting point for considering the activity of chimeric peptides made by combining peptides with different antimicrobial mechanisms. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate beta-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions.     

Molecular cartography of proteins: surface relief analysis of the calf eye lens protein gamma-crystallin

Methods of calculating the protein molecular surface and different map representations are described. The maps are obtained by projection of the space-filling molecular model on the surface of the ellipsoid of inertia. A new approach to surface analysis is proposed which is based on the use of three general maps: an identification map with all residues outlined, a surface relief map and a coloured map with a specific colour for each of the surface atoms. Superposition of these maps greatly simplifies molecular surface analysis. The usefulness of such an approach has been demonstrated by the study of the relief of the calf eye lens protein gamma-crystallin II. Protrusions of the relief have been shown to be occupied generally by charged residues, but in some cases by the hydrophobic ones. It is interesting to note that in crystal medium the protruding residues are involved, in the majority of cases, in intermolecular contacts. The protruding regions have been found to be pseudosymmetrical to each other in accordance with the two-fold rotation axis of the molecule. However, the colours of these regions, i.e. the atoms of the corresponding side chains, differ greatly.     

Heterologous expression and topography of the main intrinsic protein (MIP) from rat lens

Wild type rat lens main intrinsic protein (MIP) and MIP mutated (F73I, F75L) to resemble the glycerol facilitator of Escherichia coli in the region of the NPA1 box were used to investigate the topology of MIP in the membrane of Spodoptera frugiperda (Sf21) cells using the baculovirus expression system and expression in mouse erythroid leukaemia cells (MEL C88). Differential fixation for staining was used, with paraformaldehyde for externally exposed antigenic sites, and acetone for both externally and internally exposed protein antigenic sites. Immunofluorescence using antibodies to synthetic MIP peptides showed that wild type MIP had a six transmembrane topography. The N- and C-termini were intracellular in both expression systems, and both NPA boxes were found to be extracellular. These results show that residues around the NPA1 box can influence the folding of the MIP in the membrane, and provide structural evidence for the poor water transport properties of MIP, as the NPA boxes lie outside the plane of the membrane.