Immobilisation of antibodies in gels allows the improved release and identification of glycans.

Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodies have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high performance liquid chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. This procedure not only allowed measurement of the purity of the intact antibodies but also provided detailed analysis of the complex mixtures of oligosaccharides covalently attached to these glycoproteins. The methodology out-lined allows the simultaneous processing of a number of glycoproteins separated on one single gel. In contrast to the release of carbohydrate from glycoproteins in solution, this procedure can also be conveniently applied when only impure glycoprotein is available.

Characterization of the glycosylation profiles of Alzheimer's beta -secretase protein Asp-2 expressed in a variety of cell lines.

Amyloid 39-42 beta -peptides are the main components of amyloid plaques found in the brain of Alzheimer's disease patients. Amyloid 39-42 beta-peptide is formed from amyloid precursor protein by the sequential action of beta- and gamma-secretases. Asp-2 is a transmembrane aspartic protease expressed in the brain, shown to have beta-secretase activity. Mature Asp-2 has four N-glycosylation sites. In this report we have characterized the carbohydrate structures in this glycoprotein expressed in three different cell lines, namely Chinese hamster ovary, CV-1 origin of SV40, and baculovirus-infected SF9 cells. Biantennary and triantennary oligosaccharides of the "complex" type were released from glycoprotein expressed in the mammalian cells, whereas mannose-rich glycans were identified from glycoprotein synthesized in the baculovirus-infected cells. Site-directed mutagenesis of the asparagine residues at amino acid positions 153, 172, 223, and 354 demonstrate that the protease activity of Asp-2 is dependent on its glycosylation.

Analysis of N-linked oligosaccharides: progress towards the characterisation of glycoprotein-linked carbohydrates.

The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease.

Analysis of N-linked oligosaccharides released from glycoproteins separated by two-dimensional gel electrophoresis.

Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins (each at a level from 0.5 to 50 microg) were resolved in the first dimension according to their isoelectric points (pI), followed by separation in the orthogonal axis on the basis of their molecular weights. Glycans were released directly from excised gel spots after digestion with PNGase F, with or without prior treatment with trypsin. In a third method, glycoproteins were electroblotted onto poly(vinylidene difluoride) before glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtained after derivatization with 3-acetamido-6-aminoacridine, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to proteomics are discussed.

Phenotyping apolipoprotein E*3-leiden transgenic mice by two-dimensional polyacrylamide gel electrophoresis and mass spectrometric identification.

Apolipoprotein E (ApoE) plays an important role in cholesterol and triglyceride metabolism, being one of the major structural components of chylomicrons and very low density lipoprotein (VLDL) remnants. ApoE functions as a ligand in the receptor-mediated uptake of these remnants from the blood by the liver. A variant form of ApoE, apolipoprotein E*3-Leiden, shows reduced affinity for the low density lipoprotein (LDL) receptor, and results in the dominant expression of type III hyperlipoproteinemia. Two-dimensional electrophoresis (2-DE) has been used to characterise protein expression in serum samples from control and transgenic mice expressing the human ApoE*3-Leiden mutation, fed a cholesterol-rich diet, and transgenic mice fed a normal diet. For the identification of proteins, single silver-stained spots were excised from the 2-DE gels and subjected to in-gel enzymatic digestion. Extracted peptides were analysed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This proteomic approach has enabled the ApoE*3-Leiden variant to be positioned in a 2-DE separation of serum proteins, and has identified changes in the expression of haptoglobin, indicating that this protein may provide a marker for the potential onset of atherosclerosis.

An integrated proteomic approach to studying glomerular nephrotoxicity.

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

NADH:ubiquinone oxidoreductase from bovine heart mitochondria: sequence of a novel 17.2-kDa subunit.

The sequences of 41 subunits of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria have been described previously. Seven of them are encoded in mitochondrial DNA, and the remainder are nuclear gene products that are imported into the organelle from the cytoplasm. By electrospray mass spectrometry experiments conducted on complex I and on two related subcomplexes, an additional protein has been identified with a mass not corresponding to any of the known subunits of the enzyme. This protein has also been found in samples of the enzyme fractionated on two dimensional polyacrylamide gels. Material from these gels has been digested with trypsin and peptide sequences have been determined, confirming that the protein did not correspond to any of the known subunits of complex I. The cDNA sequence of this protein, determined with the aid of the peptide sequences, demonstrates that it is a novel subunit of complex I, and that it is related to a 13-kDa human protein associated with differentiation.

The ATPase inhibitor protein from bovine heart mitochondria: the minimal inhibitory sequence.

The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.

The F1F0-ATPase complex from bovine heart mitochondria: the molar ratio of the subunits in the stalk region linking the F1 and F0 domains.

The F1 globular catalytic domain and the F0 intrinsic membrane domain of the F1F0-ATPases in bacteria, chloroplasts, and mitochondria are connected by a slender stalk. In the F1F0 complex from bovine heart mitochondria, the stalk is thought to contain subunits OSCP, d, and F6, and the globular part of the membrane bound subunit b, referred to as b'. It has been shown previously that the OSCP, b', d, and F6 proteins can be assembled in vitro into a water soluble complex named the "stalk". The stalk and F1-ATPase together form another complex named F1.stalk. In this paper, the molar ratios of the OSCP, b (or b'), d, and F6 in the stalk, F.stalk, and F1F0-ATPase complexes have been investigated by three independent methods. By quantitation of radioactivity incorporated by S-carboxymethylation with iodo-2-[14C]acetic acid into a stalk complex containing a form of F6 with the mutation Glu3-Cys, it was shown that the stalk consists of equimolar quantities of its four constituent proteins. In the stalk complex containing the natural F6 sequence, this conclusion was confirmed both by quantitation of radioactivity incorporated by Nepsilon-acetimidation with ethyl [1-14C]acetimidate, and by quantitative N-terminal sequence analysis of subunits. By similar Nepsilon-acetimidation experiments, it has been demonstrated that the F1.stalk complex contains one copy per assembly of the OSCP, b', d, and F6 proteins and that the F1F0-ATPase contains one copy per enzyme complex of subunits OSCP, b, and d. The presence of one copy per complex of the OSCP, b' (or b), d, and F6 proteins in the F1.stalk and F1F0-ATPase complexes, respectively, was confirmed by quantitative sequencing.

The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex.

The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits.

ATP synthase from bovine heart mitochondria: identification by proteolysis of sites in F0 exposed by removal of F1 and the oligomycin-sensitivity conferral protein.

The exposure to trypsinolysis of subunits of F1F0-ATPase and of its F0 domain have been compared in everted inner membrane vesicles (submitochondrial particles) made from bovine mitochondria. Treatment of submitochondrial particles with guanidine hydrochloride removed the subunits of F1-ATPase and the oligomycin-sensitivity conferral protein (OSCP), and exposed sites that were occluded in the intact F1F0-ATPase complex. These sites were identified by purifying the subunits from the isolated F0 and F1F0-ATPase complexes before and after proteolysis of the vesicles, and by characterizing them by N-terminal sequencing and electrospray-ionization mass spectrometry. In the stripped vesicles, subunit F6 was completely digested away by either trypsin or chymotrypsin. Trypsin also cleaved subunit b, first at the bond arginine-166-glutamine-167, and then at the consecutive linkages, lysine-120-arginine-121 and arginine-121-histidine-122. Chymotrypsin-sensitive sites were observed after the adjacent methionines 164 and 165. Trypsin also removed amino acids 1-3 of subunit d, and minor cleavage sites were observed in subunit d between amino acids 24 and 25, in subunit g between amino acids 5 and 6, and after amino acid 40 in subunit e. The other subunits remained protected from proteolysis. In membrane-bound F1F0-ATPase, the N-terminus of subunit d was also accessible to trypsin, and subunit e was more susceptible to proteolysis than in F0. Otherwise the F0 subunits and the OSCP were protected. Subunits alpha and beta were cleaved by trypsin at the same sites in their N-terminal regions as in purified F1-ATPase. The trypsinized F0 was incapable of binding F1-ATPase in the presence of the OSCP. These experiments and in vitro re-assembly experiments described elsewehere, that were guided by the results of the proteolysis experiments, have helped to establish a central role for subunit b in the formation of the stalk connecting the F1 and F0 domains of the F1F0-ATPase complex.

ATP synthase from bovine heart mitochondria. In vitro assembly of a stalk complex in the presence of F1-ATPase and in its absence.

Four subunits of the F1F0-ATPase from bovine heart mitochondria have been produced by heterologous over-expression in Escherichia coli. They are the oligomycin sensitivity conferral protein (OSCP), coupling factor 6 (F6) and subunits b and d. Likewise, fragments b', bI, bC, and bM (amino acid residues 79 to 214, 121 to 214, 165 to 214 and 79 to 164, respectively, of subunit b), and fragment d' (subunit d lacking residue 1 to 14) have been produced in abundant quantities by bacterial expression. These subunits, and the fragments of subunits b and d, have been assayed singly and in various combinations by gel-filtration chromatography for their abilities to bind to bovine heart F1-ATPase. Only the OSCP was found to be capable of forming a stable binary complex with F1-ATPase. When fragments b', bI or bC were added to F1-ATPase together with the OSCP, the ternary complexes F1.OSCP.b', F1.OSCP.bI or F1.OSCP.bC were formed, but b', bI and bC appeared to be present in sub-stoichiometric amounts. When F6 was added also, then the stoichiometric quaternary complexes F1.OSCP.b'.F6 and F1.OSCP.bI.F6 were obtained, as was a fourth quaternary complex containing approximately equivalent amounts of F1 and OSCP, and sub-stoichiometric quantities of bC and F6. Finally, three pentameric complexes F1.OSCP.b'.F6.d, F1.OSCP.b'.F6.d' and F1.OSCP.b.F6.d were isolated. In a further series of reconstitution experiments, the binary complexes b'.OSCP and b'.d, the ternary complex b'.d'.F6, and the quaternary complex OSCP.b'.F6.d were obtained. The pre-formed quaternary complex produced a stoichiometric pentameric complex with F1-ATPase. It was shown by S-carboxymethylation of cysteine residues with iodo-[2-14C]acetic acid that bovine F1F0-ATPase and the reconstituted F1.stalk complex, F1.OSCP.b'.d.F6, each contained one copy per complex of subunits b (or b'), OSCP and d, and that the separate stalk complex contained the same three subunits in the approximate molar ratio 1:1:1. The ratio of b to d in purified F0 was 1:1. Finally, it was demonstrated that the binding of the various subunits to F1-ATPase increases the ATP hydrolase activity and diminishes its inactivation by exposure to cold. These assembly experiments help to define some of the inter-subunit interactions in the stalk region of the F1F0-ATPase complex, and they are an essential step forward towards the goal of extending the high-resolution structure of bovine F1-ATPase into the stalk.

Autoantibodies to lactoferrin and histone in systemic vasculitis identified by anti-myeloperoxidase solid phase assays.

We aimed at confirming the antigen specificity recognized by anti-neutrophil cytoplasm antibodies (ANCA) in patients presenting systemic vasculitis with anti-myeloperoxidase (MPO) activity on ELISA. Thirty-five consecutive patients with reactivity in anti-MPO ELISA and systemic microscopic vasculitides were tested in slot and Western blot analyses. Eleven of 35 sera exhibited binding in Western blot studies with the MPO preparation used in the ELISA: five sera bound at the size of MPO, but five sera reacted with a 78 kD species (p78) co-purifying with MPO, and one serum blotted both MPO and p78. Sequence analysis and antigen-specific assays including Western blot studies showed that p78 is lactoferrin. All anti-lactoferrin positive sera, but no anti-MPO positive sera, also exhibited anti-nuclear binding on HEp2 cells with specificity for histone. We concluded that: (a) a subgroup of patients presenting systemic vasculitis with false anti-MPO reactivity on ELISA had anti-lactoferrin antibodies; (b) anti-lactoferrin was associated with anti-nuclear activity with specificity for histone; (c) these patients had systemic vasculitis without histological evidence of immune complex deposition.

Fo membrane domain of ATP synthase from bovine heart mitochondria: purification, subunit composition, and reconstitution with F1-ATPase.

The Fo membrane domain of the F1Fo-ATP synthase complex has been purified from bovine heart mitochondria. The purification procedure involves the removal of peripheral membrane proteins, including F1-ATPase, from submitochondrial particles with guanidine hydrochloride, followed by extraction of Fo and other membrane proteins from the stripped membranes in the presence of the detergent n-dodecyl beta-D-maltoside. Fo was then purified by ion-exchange and dye ligand chromatography in the presence of the same detergent. Approximately 15 mg of pure Fo was recovered from 1.8 g of mitochondrial membrane protein. The purified Fo is a complex of nine different polypeptides. They are subunits a, b, c, d, e, F6, and A6L characterized before in F1Fo-ATPase preparations, and two new hitherto undetected subunits, named f and g. The sequences of subunits f and g have been determined. They are not related significantly to any known protein, but subunit f appears to contain a membrane-spanning alpha-helix. Proteins f and g are also present in approximately stoichiometric amounts in a highly purified preparation of intact F1Fo-ATPase, and so it is concluded that they are authentic subunits of the bovine enzyme with unknown functions. Dibutyltin 3-hydroxyflavone, an inhibitor of F1Fo-ATPase, also binds to the purified Fo in detergent and competes for binding with venturicidin. In the presence of F1 and OSCP, the purified Fo was reassembled into the intact F1Fo-ATPase complex. Therefore, this procedure provides a relatively abundant source of pure and functional Fo that is suitable for structural analysis.

Complementary DNA sequences of two 14.5 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Completion of the primary structure of the complex?

The amino acid sequences of two nuclear-encoded subunits of complex I from bovine heart mitochondria have been determined. Both proteins have an apparent molecular weight of 14.5 kDa and their N-alpha-amino groups are acetylated. They are known as subunits B14.5a and B14.5b. Neither protein is evidently related to any known protein and their functions are obscure. A total of 34 nuclear-encoded subunits of bovine complex I have now been sequenced and it is thought that the primary structure of the complex is now complete, although with such a complicated structure it is difficult to be certain that there are no other subunits remaining to be sequenced. Seven additional hydrophobic subunits of the enzyme are encoded in mitochondrial DNA, and therefore bovine heart complex I is an assembly of about 41 different proteins. If it is assumed that there is one copy of each protein in the assembly, these polypeptides contain 7,955 amino acids in their sequences, more than are found in the Escherichia coli ribosome, which contains 7,336 amino acids in its 32 polypeptides.

Resolution of NADH:ubiquinone oxidoreductase from bovine heart mitochondria into two subcomplexes, one of which contains the redox centers of the enzyme.

NADH:ubiquinone oxidoreductase (complex I) was purified from bovine heart mitochondria by solubilization with n-dodecyl beta-D-maltoside (lauryl maltoside), ammonium sulfate fractionation, and chromatography on Mono Q in the presence of the detergent. Its subunit composition was very similar to complex I purified by conventional means. Complex I was dissociated in the presence of N,N-dimethyldodecylamine N-oxide and beta-mercaptoethanol, and two subcomplexes, I alpha and I beta, were isolated by chromatography. Subcomplex I alpha catalyzes electron transfer from NADH to ubiquinone-1. It is composed of about 22 different and mostly hydrophilic subunits and contains 2.0 nmol of FMN/mg of protein. Among its subunits is the 51-kDa subunit, which binds FMN and NADH and probably contains a [4Fe-4S] cluster also. Three other potential Fe-S proteins, the 75- and 24-kDa subunits and a 23-kDa subunit (N-terminal sequence TYKY), are also present. All of the Fe-S clusters detectable by EPR in complex I, including cluster 2, are found in subcomplex I alpha. The line shapes of the EPR spectra of the Fe-S clusters are slightly broadened relative to spectra measured on complex I purified by conventional means, and the quinone reductase activity is insensitive to rotenone. Similar changes were found in samples of the intact chromatographically purified complex I, or in complex I prepared by the conventional method and then subjected to chromatography in the presence of lauryl maltoside. Subcomplex I beta contains about 15 different subunits. The sequences of many of them contain hydrophobic segments that could be membrane spanning, including at least two mitochondrial gene products, ND4 and ND5. The role of subcomplex I beta in the intact complex remains to be elucidated.

Sequences of 20 subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria. Application of a novel strategy for sequencing proteins using the polymerase chain reaction.

NADH:ubiquinone oxidoreductase, the first enzyme in the respiratory electron transport chain of mitochondria, is a membrane-bound multi-subunit assembly, and the bovine heart enzyme is now known to contain about 40 different polypeptides. Seven of them are encoded in the mitochondrial DNA; the remainder are the products of nuclear genes and are imported into the organelle. The primary structures of 12 of the nuclear coded subunits have been described and those of a further 20 are described here. The subunits have been sequenced by following a strategy based on the polymerase chain reaction. This strategy has been tailored from existing methods with the twofold aim of avoiding the use of cDNA libraries, and of obtaining a cDNA sequence rapidly with minimal knowledge of protein sequence, such as can be determined in a single N-terminal sequence experiment on a polypeptide spot on a two-dimensional gel. The utility and speed of this strategy have been demonstrated by sequencing cDNAs encoding 32 nuclear-coded-membrane associated proteins found in bovine heart mitochondria, and the procedures employed are illustrated with reference to the cDNA sequence of the 20 subunits of NADH:ubiquinone oxidoreductase that are presented. Extensive use has also been made of electrospray mass spectrometry to measure molecular masses of the purified subunits. This has corroborated the protein sequences of subunits with unmodified N terminals, and their measured molecular masses agree closely with those calculated from the protein sequences. Nine of the subunits, B8, B9, B12, B13, B14, B15, B17, B18 and B22 have modified alpha-amino groups. The measured molecular masses of subunits B8, B13, B14 and B17 are consistent with the post-translational removal of the initiator methionine and N-acetylation of the adjacent amino acid. The initiator methionine of subunit B18 has been removed and the N-terminal glycine modified by myristoylation. Subunits B9 and B12 appear to have N-terminal and other modifications of a hitherto unknown nature. The sequences of the subunits of bovine complex I provide important clues about the location of iron-sulphur clusters and substrate and cofactor binding sites, and give valuable information about the topology of the complex. No function has been ascribed to many of the subunits, but some of the sequences indicate the presence of hitherto unsuspected biochemical functions. Most notably the identification of an acyl carrier protein in both the bovine and Neurospora crassa complexes provides evidence that part of the complex may play a role in fatty acid biosynthesis in the organelle, possibly in the formation of cardiolipin.(ABSTRACT TRUNCATED AT 400 WORDS)