Deimination of myelin basic protein. 2. Effect of methylation of MBP on its deimination by peptidylarginine deiminase

Deimination of myelin basic protein (MBP) has been implicated in the chemical pathogenesis of multiple sclerosis (MS). Degradation of bovine MBP by cathepsin D, a myelin-associated protease, was increased when 6 arginyl residues were deiminated and became very rapid when all 18 arginyl residues were deiminated. Since MBP contains a number of modifications, including methylation, phosphorylation, etc., we studied the effect of methylation, an irreversible modification, to determine how this modification affected deimination. Methylation of Arg 106 in bovine MBP (Arg 107 in human), a naturally occurring modification of MBP, has been shown to affect the deimination of arginyl residues in the present study. Since fractionation of MBP into unmethylated, monomethylated, and dimethylated species cannot be done readily on a preparative scale, mass spectrometry with the Q-TOF instrument resolved these species readily since each differed from the other by 14 atomic mass units (amu). Examination of five different hMBP samples, two from normal brain and three from MS brain, revealed that increased deimination of arginyl residues correlated with a decreased methylation of Arg 107 (human sequence). To study this process in vitro, bovine MBP (bMBP) was used. Component 1 (C-1) is the most cationic of the MBP "charge isomers" and the most unmodified, in which all arginyl residues are intact. It was deiminated to various extents with purified bovine brain peptidylarginine deiminase, generating a number of species containing 0-13.7 mol of citrulline/mol of bMBP. Mass spectrometry of each of these species permitted us to determine the influence of methylation of Arg 106 (bovine sequence) on deimination by this enzyme. We found that bMBP with unmethylated arginine was deiminated at a rate of 0.081 mol of citrulline/min, with monomethylarginine, 0.068 mol of citrulline/min, and with dimethylarginine, 0.036 mol of citrulline/min. We suggest that the methylated arginyl residue becomes sequestered in the hydrophobic beta-sheet structure and disrupts the three-dimensional structure of the protein so that other arginyl residues are less accessible to peptidylarginine deiminase.     

A heme binding site on myelin basic protein: characterization, location, and significance

Myelin basic protein (MBP), an extrinsic membrane protein from the myelin sheath, binds dicyanohemin. The binding generates absorption bands in the Soret region and quenches the fluorescence emitted by the sole tryptophan residue. The absorption titration curves in the Soret demonstrate that the binding is stoichiometric, one heme per protein, with a large value of the extinction coefficient (8 X 10(4) M-1 cm-1 at 420 nm). Fluorescence quenching titration curves indicate an identical stoichiometry and a low quenching efficiency of 20%. From the heme titration curve the association constant between dicyanohemin and MBP is estimated to be greater than or equal to 10 nM-1 in 50 mM 4-morpholinepropanesulfonic acid buffer, pH 7.0, at 20 degrees C. Digestion of MBP by Staphylococcus aureus V8 protease yields a peptide (38-118) whose heme binding properties are identical to those of MBP. In contrast, peptides obtained by digestion of MBP with cathepsin D do not exhibit any specific binding of dicyanohemin. The cleavage of the Phe-Phe (42-43) bond appears to be critical in this respect. A comparison of the sequence immediately preceding, including these residues with a probable heme binding site of a mitochondrial cytochrome b, reveals a high degree of homology. The possible significance of heme binding is discussed.     

An Arg/Lys-->Gln mutant of recombinant murine myelin basic protein as a mimic of the deiminated form implicated in multiple sclerosis

The degree of post-translational enzymatic deimination (conversion of arginyl to citrullinyl residues) of myelin basic protein (MBP) is correlated with the severity of the human autoimmune disease multiple sclerosis (MS). It is difficult to obtain large quantities of deiminated MBP from natural sources (autopsy material), and in vitro deimination using peptidylarginine deiminase (EC 3.5.3.15) is both non-specific and irreproducible. Since there is no known codon for citrulline, we have constructed a mutant form of recombinant murine MBP (rmMBP) in which 5 Arg and 1 Lys residues have been replaced by Gln as the most reasonable analogue of Cit. The residues were chosen to correspond to the 6 Arg residues in human MBP which are most commonly deiminated in chronic MS. The mutant species, rmMBP-qCit(6) where the "q" represents "quasi-," was probed by numerous biochemical and biophysical techniques. Highly homogeneous protein preparations were obtained using a modified expression system which minimised spurious misincorporation of Lys for Arg, as ascertained by electrospray ionisation mass spectrometry. The mutant form rmMBP-qCit(6) had a reduced ability to aggregate lipid vesicles, a slightly greater susceptibility to digestion by cathepsin D, a greater proportion of random secondary structure, and different conformational responses to lipids, compared with the unmodified rmMBP. Overall, the mutant protein's properties were consistent with the effects of deimination and support its use as a model for evaluating the effects of this modification.     

Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein.

Myelin basic protein (MBP) exists in a population of isoforms and isomers. The 18.5 kDa MBP-C1, the main human adult isoform, has 170 residues and is relatively unmodified, whereas the same isoform can be citrullinated on six arginine residues to create the MBP-C8 (MBP Cit6) isomer. MBP Cit6 dominates in MS brain, accounting for 45% rather than 25% of the population of MBP isomers. In the fulminant form of MS, known as Marburg's Disease, 18 of the 19 arginines in MBP are citrullinated (MBP Cit18). Citrullination of MBP could lead to instability of myelin or limited remyelination. In this investigation, the susceptibilities to degradation by cathepsin D of MBP Cit6 and MBP-C1, both from normal and MS brain tissue, and Marburg MBP Cit18 were compared. The pattern of digestion was similar, and no differences of corresponding isomers in normal and MS brain were noted. However, normal MBP Cit6 was degraded 10-fold more rapidly than MBP-C1, and MBP Cit18 was degraded even more rapidly. MBP peptide 45-89 was preserved regardless of isomer type or source. Its generation was directly related to the citrulline content of the MBP substrate being 4 times faster in normal MBP Cit6 and 35 times faster in Marburg MBP Cit18 than in normal MBP-C1. Peptide 45-89 from a citrullinated MBP exhibited more deamidation, and, regardless of source, showed an alpha-helix structure in a lipid mimetic environment. We postulate that the generation of MBP peptides, including those that are dominant and encephalitogenic, is directly related to deimination of arginine to citrulline in MBP.     

Interactions of the 18.5-kDa isoform of myelin basic protein with Ca(2+)-calmodulin: in vitro studies using fluorescence microscopy and spectroscopy.

The interactions of the 18.5-kDa isoform of myelin basic protein (MBP) with calmodulin (CaM) in vitro have been investigated using fluorescence microscopy and spectroscopy. Two forms of MBP were used: the natural bovine C1 charge isomer (bMBP/C1) and a hexahistidine-tagged recombinant murine product (rmMBP), with only minor differences in behaviour being observed. Fragments of each protein generated by digestion with cathepsin D (EC 3.4.23.5) were also evaluated. Using fluorescence microscopy, it was shown that MBP and CaM interacted in the presence of Ca2+ under a variety of conditions, including high urea and salt concentrations, indicating that the interaction was specific and not merely electrostatic in nature. Using cathepsin D digestion fragments of MBP, it was further shown that the carboxyl-terminal domain of MBP interacted with Ca(2+)-CaM, consistent with our theoretical prediction. Spectroscopy of the intrinsic fluorescence of the sole Trp residue of MBP showed that binding was cooperative in nature. The dissociation constants for formation of a 1:1 MBP-Ca(2+)-CaM complex were determined to be 2.1 +/- 0.1 and 2.0 +/- 0.2 microM for bMBP/C1 and rmMBP, respectively. Fluorescence spectroscopy using cathepsin D digestion fragments indicated also that the carboxyl-terminal region of each protein interacted with Ca(2+)-CaM, with dissociation constants of 1.8 +/- 0.2 and 2.8 +/- 0.9 microM for the bMBP/C1 and rmMBP fragments, respectively. These values show a roughly 1000-fold lower affinity of MBP for CaM than other CaM-binding peptides, such as myristoylated alanine-rich C-kinase substrate, that are involved in signal transduction.     

Phosphorylation of myelin basic protein by glycogen phosphorylase kinase

The ability of homogeneous glycogen phosphorylase kinase (Phk) from rabbit skeletal muscle to phosphorylate bovine brain myelin basic protein (MBP) was investigated. Phk could incorporate a maximum of 1.9 mol phosphate/mol MBP. The apparent Km and Vmax for Phk phosphorylation of MBP were 27 microM and 90 nmol/min per mg enzyme, respectively. Properties of MBP phosphorylation by Phk are similar to those of phosphorylase as a substrate. Only serine residues of MBP are phosphorylated by Phk. Phosphorylation sites of MBP by Phk are not identical to those by cAMP-dependent protein kinases.     

The preparation of antibodies reactive against citrulline-containing charge isomers of myelin basic protein but not against the arginine-containing charge isomers.

Human myelin basic protein (MBP) is composed of several charge isomers, the result of various post-translational modifications. One of the charge isomers C-8, has been shown in our laboratory to contain six citrullinyl residues which replace arginyl residues at selected sites in the MBP. In order to determine the disposition of the citrulline-containing charge isomers in the myelin stack, we prepared specific antisera against the citrullinyl group. Since 9-fluorenylmethoxycarbonyl (Fmoc)-citrulline, required for the preparation of the synthetic peptides to be used for antibody production, was not commercially available, synthesis of the Fmoc-citrulline was a necessary prerequisite. The synthesis and purification of the N-9-fluorenylmethyloxycarbonyl derivative of citrulline is described. It was characterized by thin layer chromatography, 1H and 13C NMR spectroscopy, fast-atom bombardment mass spectroscopy, and thermal analyses. It was used in the automated peptide synthesis of a peptide Ala-Cit-His-Gly-Phe-Leu-Pro-Cit-His-Arg corresponding to residues 24-33 and Gly-Cit-Asp-Ser-Arg-Ser-Gly-Ser-Pro-Met-Ala-Cit-Arg, corresponding to residues 158-170 of the C-8 sequence, a naturally occurring charge isomer of human myelin basic protein, and a tetracitrulline peptide, Cit-Cit-Cit-Cit-Gly. The tetracitrulline peptide was used for the production of an antibody shown to react only with synthetic peptides and proteins containing citrulline. This antibody was used to distinguish between a citrulline-containing protein, C-8, a naturally occurring charge isomer of MBP, and a non-citrulline-containing charge isomer of MBP, C-1.     

Primary structure of equine myelin basic protein by mass spectrometry

Equine myelin basic protein (MBP) has been isolated from spinal cord and shown to consist of a number of components (charge isomers) by alkaline-urea gel electrophoresis. Mass analyses of several of these components showed that each was posttranslationally modified and some have been identified. Component 1, the most cationic charge isomer, was sequenced by a combination of liquid chromatography and mass spectrometry of peptides obtained by proteolytic digestion. At 172 residues it is slightly larger than the bovine (169) and the human (170). A major difference between bovine and equine sequences was the replacement of AQGH (bovine residues 76-79) by SRDG (equine). A number of other replacements involving single amino acids were also found. Methylated arginine (residue 108 equine) was found as both the mono- and the dimethylated derivative and represents the first MS/MS evidence for this modification in any MBP.     

Role of phosphorylation in conformational adaptability of bovine myelin basic protein.

Controlled thrombic digestion of a preparation of components 2 + 3 isolated from the 18.5 kDa bovine myelin basic protein (MBP) yielded a polypeptide that was monophosphorylated on threonine 97 (component 3pT97). This is the first posttranslationally phosphorylated MBP isolated in pure form. We studied the effect of this single phosphate on the conformational adaptability of 18.5 kDa bovine MBP by comparing the circular dichroism (CD) spectrum of component 3pT97 with the spectra of highly purified nonphosphorylated components 1 and 2. The CD spectra of nonphosphorylated component 1 and component 2 [monodeamidated form(s) of component 1] were indistinguishable, while component 3pt97 exhibited a different spectrum. The singly phosphorylated MBP component exhibited 13% more ordered conformations than that adopted by nonphosphorylated MBP in dilute aqueous solutions. This was estimated from the CD spectra, and apparently involved about 17 additional amino acid residues in beta-structure(s).     

The isolation, characterization, and lipid-aggregating properties of a citrulline containing myelin basic protein

Human myelin basic protein was fractionated into its various charge isomers by CM52 cation exchange chromatography. Approximately 25-30% of the total charge applied to the column appeared in the void volume. This material termed "C-8," was further purified by reversed phase high performance liquid chromatography. Amino acid analyses of C-8 revealed low Arg (7 residue % in C-8 compared to 11-12 residue % in C-1) and increased Glx residues. The low Arg was accounted for by a corresponding amount of citrulline. Sequence analysis after chemical fragmentation (cyanogen bromide and BNPS-skatole) and enzymatic (cathepsin D and carboxypeptidase S-1) digestion localized the citrulline at residues 25, 31, 122, 130, 159, and 170 of the amino acid sequence. The effect of this loss of positive charge on the ability of the protein to aggregate lipid vesicles was demonstrated with vesicles composed of phosphatidylcholine (92.2 mol %) and phosphatidylserine (7.8 mol %). C-1 was the most effective charge isomer, and C-8 was the least effective. The ability of these charge isomers to aggregate vesicles correlated with the net positive charge on each. Vesicles composed of phosphatidylcholine alone were not aggregated by lipophilin or any of the charge isomers. However, when lipophilin was incorporated into phosphatidylcholine vesicles (50% w/w), small, optically clear suspensions of vesicles were formed. None of C-1, C-2, or C-3 aggregated these vesicles, but C-8 produced rapid vesicle aggregation. Since the substitution of citrulline for Arg would generate several relatively long apolar sequences, these would enhance the ability of C-8 to interact with the hydrophobic lipophilin molecule, promoting vesicle aggregation by hydrophobic interactions. The mechanism by which citrulline is generated in myelin is not known, although enzymatic conversion has been described in other systems. Studies are underway to elucidate the mechanism by which this post-translational modification is generated.     

Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues.

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.     

Adduction of cholesterol 5,6-secosterol aldehyde to membrane-bound myelin basic protein exposes an immunodominant epitope

Myelin degradation in the central nervous system (CNS) is a clinical hallmark of multiple sclerosis (MS). A reduction in the net positive charge of myelin basic protein (MBP) via deimination of arginine to citrulline has been shown to correlate strongly with disease severity and has been linked to myelin instability and a defect that precedes neurodegeneration and leads to autoimmune attack. Recently, we have shown that lipid-derived aldehydes, such as cholesterol 5,6-secosterols atheronal A (1a) and atheronal B (1b), modulate the misfolding of certain proteins such as apolipoprotein B(100), β-amyloid, α-synuclein, and κ- and λ-antibody light chains in a process involving adduction of the hydrophobic aldehyde to lysine side chains, resulting in a decrease in the net positive charge of the protein. In this study, we show that the presence of either atheronal A (1a) or atheronal B (1b) in large unilamellar vesicles (cyt-LUVs) with the lipid composition found in the cytosolic myelin sheath and bovine MBP (bMBP) leads to an atheronal concentration-dependent increase in the surface exposure of the immunodominant epitope (V86-T98) as determined by antibody binding. Other structural changes in bMBP were also observed; specifically, 1a and 1b induce a decrease in the surface exposure of L36-P50 relative to control cyt-LUVs as measured both by antibody binding and by a reduction in the level of cathepsin D proteolysis of F42 and F43. Structure-activity relationship studies with analogues of 1a and 1b point to the aldehyde moiety of both compounds being critical to their effects on bMBP structure. The atheronals also cause a reduction in the size of the bMBP-cyt-LUV aggregates, as determined by fluorescence microscopy and dynamic light scattering. These results suggest that formation of an imine between inflammatory-derived aldehydes, which effectively reduces the cationic nature of MBP, can lead to structural changes in MBP and a decrease in myelin stability akin to deimination and as such may make a hitherto unknown contribution to the onset and progression of MS.     

Phosphorylation of myelin basic protein and peptides by ganglioside-stimulated protein kinase

Rabbit myelin basic protein (MBP) was phosphorylated by a ganglioside-stimulated protein kinase to a stoichiometry of 1.4 and 2.1 mol phosphate/mol MBP in the presence and absence of GTlb, respectively. Two-dimensional peptide mapping analyses revealed that two of the sites of phosphorylation were distinct from those catalyzed by cAMP-dependent protein kinase or protein kinase C. Phosphorylation of one of these sites by ganglioside-stimulated protein kinase was inhibited by GTlb, suggesting that the inhibitory effect of gangliosides on MBP phosphorylation may be substrate-directed. Although ganglioside-stimulated protein kinase did not phosphorylate MBP at a domain containing residues 82-117, a synthetic peptide Arg-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys corresponding to residues 111-120 was phosphorylated by the kinase in a ganglioside-stimulated manner. These findings suggest that the conformation of MBP may be important in determining its phosphorylatability.     

Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein

Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out.     

Interaction of glycosylated human myelin basic protein with lipid bilayers

Myelin basic protein (MBP), isolated from normal human myelin, was glycosylated with UDP-N-acetyl-D-galactosamine and a glycosyltransferase isolated from porcine submaxillary glands. MBP containing 0.85 mol of N-acetyl-D-galactosamine per mole of protein was oxidized at carbon 6 by galactose oxidase and complexed with a spin-label, Tempoamine, in order to study its interactions with lipids. When the spin-labeled MBP was reacted with lipid vesicles consisting of DSPG, DPPG, and DMPG, most of the spin-label was motionally restricted in the gel phase, with a correlation time greater than 10(-8)s. The motion increased with increasing temperature and was sensitive to the lipid phase transition. Interaction with the gel phase of DPPA caused much less motional restriction of the probe. However, melting of the lipid allowed increased interaction and motional restriction of the probe, which was only partially reversed on cooling back to the gel phase. The motional restriction of the probe in these lipids is attributed to its penetration partway into the lipid bilayer in both the gel and liquid-crystalline phases. The fact that the probe bound to the protein can penetrate partway into the bilayer suggests that other hydrophobic side chains and residues of the protein can similarly penetrate into the bilayer. Additional evidence for penetration was provided by digestion of the lipid-bound protein with endoproteinase Lys-C. When nonglycosylated and glycosylated MBP in solution was treated with Lys-C, extensive digestion occurred. A single radioactive peptide which eluted at 25 min was identified as residues 92-105.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity for myelin basic protein-specific protein methylase I.

The substrate specificity of bovine brain myelin basic protein (MBP)-specific protein methylase I (S-adenosyl-L-methionine:protein-L-arginine N-methyltransferase, EC 2.1.1.23), which methylates arginine residues of protein, has been studied using various MBPs, several synthetic peptides and heterogeneous nuclear ribonucleoprotein complex protein (hnRNP). (1) Among MBPs from different species of brain, the carp MBP was found to be the best substrate for MBP-specific protein methylase I. This high degree of methyl acceptability is most likely due to the fact that carp MBP is not in vivo methylated at the arginine residue (Deibler, G.E. and Martenson, R.E. (1973) J. Biol. Chem. 248, 2387-2391) and that the methylatable amino acid sequence is present in this protein. (2) In order to study the minimum chain length of MBP polypeptide which functions as the methyl acceptor, several synthetic polypeptides whose sequences are identical to the region surrounding the residue 107 of bovine MBP (the in vivo methylation site) were synthesized. It was found that the hexapeptide, Gly-Lys-Gly-Arg-Gly-Leu (corresponding to residues 104-109 of bovine MBP), was the shortest methyl accepting peptide, while the tetrapeptide, Gly-Arg-Gly-Leu (corresponding to residues 106-109) was inactive as a substrate. (3) hnRNP protein is known to contain methylarginine at residue 193 (Williams, K.R., Stone, K.L., LoPresti, M.B., Merrill, B.M. and Plank, S.R. (1985) Proc. Natl. Acad. Sci. USA 82, 5666-5670) which is post-translationally modified. Thus, the RNP protein overproduced in Escherichia coli and therefore did not contain methylarginine was examined for its methyl acceptability. It was found that neither MBP-specific nor histone-specific protein methylase I could methylate this methylarginine-less RNP protein. This suggests a possible existence of a distinct protein methylase I specific for this nuclear protein.     

Large peptides of bovine and guinea pig myelin basic proteins produced by limited peptic hydrolysis.

Bovine and guinea pig myelin basic proteins were cleaved with pepsin at pH 3.0 or pH 6.0 (enzyme/substrate, 1:500, w/w), and the peptides were isolated and identified. At pH 3.0 cleavage of the bovine protein occurred principally at three sites: Phe-Phe (88-89), Phe-Phe (42-43), and Leu-Asp (36-37). Minor cleavages occurred at Leu-Ser (110-111), Phe-Ser (113-114), and Ile-Phe (152-153). A study of the time course of the hydrolysis showed that the reaction was biphasic; nearly all of the protein was cleaved at Phe-Phe (88-89) before significant cleavages at other sites occurred. At pH 6.0 cleavage of the bovine protein occurred almost exclusively at a single site, the Phe-Phe bond at position 88-89, resulting in bisection of the protein. Treatment of the guinea pig protein with pepsin under the same conditions resulted in the production of peptides which were identical with those of the bovine protein in chromatographic and electrophoretic properties and in N-terminal and C-terminal residues but which differed slightly in amino acid composition.     

The association of myelin basic protein with itself and other proteins

Chromatographic studies were performed to measure myelin basic protein (MBP) interactions by covalently binding a number of different proteins to Sepharose and passing radioactive bovine MBP over these columns. Studies at a variety of pH values, ionic strengths and temperatures revealed that the bovine MBP could interact with itself as well as cytochrome c, lysozyme, and ovalbumin. Chromatographic profiles of elution volume vs. pH revealed that the interaction between MBP and these immobilized proteins was biphasic. The self-association of MBP was found to be strongest between pH 7.4 and 8.1 and at an elevated temperature. Titration of the amino acid residues responsible for the association of MBP with other proteins revealed apparent pKs ranging from 6.10 to 6.70. A pH dependence study at an elevated temperature shifted the apparent pK of the MBP interaction to a lower value with all the proteins except ovalbumin. After destroying 60% of the histidine residues in MBP by photooxidation and passing¹²⁵I-labeled photooxidized MBP over Sepharose columns containing immobilized protein, the second phase in binding was decreased significantly with immobilized cytochrome c, lysozyme, and MBP and to a smaller extent with ovalbumin. These results are consistent with the involvement of deprotonated histidine residues in the MBP-protein associations.     

The influence of pH on the degradation of bovine myelin basic protein by bovine brain cathepsin

The degradation of bovine myelin basic protein by bovine brain cathepsin D (ED 3.4.23.5) was studied over a pH range of 2.75 - 6.0. Throughout this pH range pepstatin, an inhibitor of cathepsin D, prevented the degradation. The degradation at a pH away from the optimum of pH 3.5 was predictably slower, but also resulted in more restricted cleavage. Above pH 4.5 bovine basic protein peptide 1 - 42 was not degraded further to peptide 1 - 36 as occurs at pH 3.5. Additionally, at pH 5.5 another fragment of basic protein, peptide 1 - 91, persisted indicating that under certain basic protein as well as basic protein peptide 43 - 169 may be cleaved in the molecular region of basic protein around the phenylalanyl-phenylalanine residues at position 88 - 89. The small amount of peptides 1 - 91 and 92 - 169 detected at pH 5.5 suggests that the bond between residues 91 and 92 in intact basic protein is a minor cleavage site. The options and variation in cleavage around residues 88 - 92 of basic protein presumably result from pH-dependent changes in conformation in the is region but could also be due to changes in conformation of cathepsin D. These results indicate that local tissue changes such a pH amy affect not only the velocity of the reaction but also the nature of th product formed by the degradation of basic protein by brain cathepsin D     

Mechanism of the interaction between myelin basic protein and the myelin membrane; the role of arginine methylation

The addition of solutions of bovine myelin basic protein to suspensions of unilamellar vesicles prepared from whole myelin suspensions results in the rapid equilibrium association of the vesicles into dimers, followed by time-dependent aggregation reactions. Other cationic proteins also induce the dimerization of the vesicles and equilibrium constants for dimer formation are obtained for bovine myelin basic protein, lysozyme, polyhistidine and myelin basic protein from carp, which differs from the bovine protein in that it contains no methylarginine residues. The bovine protein is more efficient at inducing dimer formation than the carp protein by approximately 0.93 kcal/mole; the carp protein is approximately as effective as the other cationic proteins examined. Complete methylation of the bovine MBP by AdoMet:MBP methyltransferase increases the interaction between MBP and the membrane by approximately 0.13 kcal/mole, consistent with the suggestion that a large portion of the free energy difference between the carp and bovine proteins arises from favorable interactions involving the methylarginine residues.