Human Myelin Basic Protein Peptide Research Articles

Diversity of T cell receptor alpha and beta chain genes expressed by human T cells specific for similar myelin basic protein peptide/major histocompatibility complexes.

T cell receptor (TcR) alpha and beta nucleotide sequences involved in the human autoreactivity to myelin basic protein (MBP) were studied by screening cDNA libraries derived from 11 independent T lymphocyte clones (TCC) established from multiple sclerosis patients and healthy donors. The TCC with defined MBP peptide specificity and HLA-DR restriction expressed multiple TcR. Even TCC recognizing the same human MBP peptide [amino acids (aa) 139-153] in identical or very similar HLA-DR context expressed diverse TcR. Two TCC which recognized peptide aa 139-153 equally well in the context of both HLA-DR2a and -DR1 molecules used distinct TcR alpha but identical beta chains. The knowledge of TcR beta and TcR alpha chain sequences of human MBP-specific T cells will allow studies correlating structure and function of TcR and their targets in MBP autoreactivity. This may have an impact on the development of immunotherapies in multiple sclerosis.

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referres to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1–14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10–19 and 90–170, but not to MBP peptide 69–89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10–19 but not for anti-MBP 1–9 or anti-MBP 80–89. These findings imply differences in sequence or conformation in the structure of MBP-C8 compared to MBP-C1, most notably near the amino terminus.

An idiotype shared by monoclonal antibodies to different peptides of human myelin basic protein.

A mAb of the IgG1/kappa isotype was raised against human myelin basic protein (MBP) peptide acetyl 1-9. This mAb, termed F23, reacted with human MBP and human MBP peptides acetyl 1-9, 1-14, and 1-44, but not with MBP peptides 10-19, 80-89, or 45-89. According to the guidelines of the molecular recognition theory, a complementary peptide to human MBP peptide 1-9 was synthesized and used to raise murine mAb with anti-Id activity. Two mAb anti-Id, F25F7 and F25C8, both of the IgM/kappa isotype, were selected for further study. These anti-Id reacted with F23, the mAb for which they were selected, and also reacted with another mAb, which was of the IgG1/kappa isotype and was raised to human MBP peptide 80-89. There was no reaction with another control mAb of the IgG1/kappa isotype or murine myeloma IgG1. By immunoblotting techniques, it was demonstrated that the Id on each of the mAbs to MBP peptides was located on the kappa L chain but also could be recognized in nonreduced IgG. The cross-reactive anti-Id suppressed antibody secretion of Id-producing hybridoma cells in an Id-specific manner, and kinetic studies suggest an intracellular mechanism for the suppression. These cross-reactive Id among antibodies to different MBP peptides imply that the same V region genes of kappa L chains are involved in the selection of antibodies to an autoantigen, like MBP, and may play a role in the modulation of immune responses against MBP in certain inflammatory demyelinating diseases.

The modulatory effect of anti-idotypic antibody on hybridoma cells secreting antibody to human myelin basic protein peptide 80–89

The idiotype (ID) of an antibody is postulated to be involved in immunoregulation by means of the immune network. This hypothesis was examined by stuying the effect of a monoclonal antibody (MAb) anti-ID on hybridoma cells secreting an ID-bearing MAb, 845D3, to human myelin basic protein (MBP) peptide 80–90. The monoclonal anti-ID (IgM/kappa), reacted with heavy and light chains of 845D3, an IgG 1/kappa MAb, but not with a control MAb of the same isotype specific for an MBP peptide differing by one amino acid residue. Fluorescence-activated cell sorting revealed IgG heavy and kappa light chains on the surface and in the cytoplasm of 90% or more of the 845D3 cells. The anti-ID did not react with the surface of the 845D3-secreting cells, and reacted with the cytoplasm of 14–18% of these cells. In spite of the absence of cell surface ID, the anti-ID significantly decreased antibody production by the ID-secreting cells. There was no effect of the anti-ID on control hybridoma cells secreting MAb to another MBP peptide. Anti-ID exerted no cytotoxic effect on ID-bearing hybridoma cells and in fact caused a marginal increase in their proliferation compared to control MAb. These results indicate that an anti-ID may alter the antibody production of ID-secreting cells in a specific manner and without cytotoxicity. This may be one of the means for controlling an immune response against MBP generated by the cells of the immune system or in situ within the nervous system. The cellular mechanism(s) for this effect remains to be defined.

Conformational Correlates of the Epitopes of Human Myelin Basic Protein Peptide 80–89

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.

Monoclonal idiotypic and anti-idiotypic antibodies produced by immunization with peptides specified by a region of human myelin basic protein mRNA and its complement

Murine monoclonal antibodies (MAbs) selective for an idiotope on a monoclonal antibody (IgG 1/kappa) to human myelin basic protein (MBP) peptide 80–89 were prepared by immunization with a synthetic decapeptide specified by RNA that is complementary to the mRNA for human MBP peptide 80–89. The monoclonal anti-idiotypic antibody (anti-ID) reacted with the MAb to human MBP peptide 80–89 but not with a MAb to bovine MBP peptide 79–88 or to murine myeloma IgG 1. The reaction between the monoclonal anti-ID and the MAb to the human MBP peptide 80–89 could be inhibited by human MBP peptide 80–89 and to a more limited degree with human MBP peptide 76–85 and bovine MBP peptide 79–88, but not by human MBP peptides 69–81 and 85–96. Practically, the use of a complementary peptide for stimulating an anti-ID response permits a more selective and feasible method for peparing anti-ID reagents. Theoretically, these results provide further support for the molecular basis of the network hypothesis.

Human myelin basic protein peptide 69–89: Immunochemical features and use in immunoassays of cerebrospinal fluid

The characteristics and heterogeneity of the myelin basic protein (MBP)-like material appearing in cerebrospinal fluid (CSF) after acute central nervou s system (CNS) myelin injury are unresolved. Antigenic material containing an epitope in the carboxyl-terminal portion of human MBP peptide 45–89 (from the intact molecule of 170 residues) is a prominent species of the MBP-like material present. In an effort to define further the MBP-like material in CSF and to enhance its detection, a modified double-antibody radioimmunoassay has been developed using a radio-ligand of human MBP synthetic peptide 69–89. This assay is more sensitive with results paralleling those of previously used MBP assays for CNS myelin damage. Results with this assay provide additional confirmation of the presence of an epitope of MBP in the decapeptide of MBP 80–89 but in a conformation simulating that of intact MBP in CSF after CNS myelin injury. Unexpected buffer effects were noted to influence the immunochemical behavior of some of the small peptides of MBP.

The presence of immunoreactive myelin basic protein peptide in urine of persons with multiple sclerosis

A polyclonal antiserum has been produced that can detect nanogram amounts of myelin basic protein (MBP)-like material in unconcentrated human urine. The urinary immunoreactive material is cross-reactive with human MBP peptides 45-89 and 69-89, dialyzable, heat resistant, and is not artifact of either degradation of radioligand or salt effect. An octapeptide, MBP peptide 82-89, was demonstrated to be the smallest peptide containing the main epitope against which this antiserum was directed. This epitope differed from the major epitope recognized by antisera detecting MBP-like material in cerebrospinal fluid, implying that the MBP-like material is altered, presumably degraded, in the kidney. Results of gel filtration and high-performance liquid chromatography suggested a size of 1,000 daltons or less and a charge similar to that of human MBP peptide 80-89. In a group of 39 persons with multiple sclerosis, 48 with other neurological diseases, and 26 normal control subjects, the concentration of urinary MBP-like material, related to the concentration of urinary creatinine, was significantly higher in the multiple sclerosis group (22.0 ng MBP-like material/mg creatinine) than in the other neurological diseases or control groups, in which the values were 7.0 and 3.9 ng MBP-like material/mg creatinine, respectively. Variations in the level of MBP-like material appearing in the urine may provide a clinically feasible test for myelin damage. The precise identification of the chemical nature of the urinary MBP-like material may also furnish a means for further analyzing the in vivo catabolism of the potentially autoantigenic MBP.

Epitopes of immunoreactive myelin basic protein in human cerebrospinal fluid

To define in more detail the features of the immunoreactive myelin basic protein (MBP) present in cerebrospinal fluid (CSF) of humans following acute injury to central nervous system myelin, the epitopes of MBP recognized by three different antisera, each capable of detecting immunoreactive MBP in CSF, were examined. All three antisera reacted well with human MBP and human MBP peptide 45-89. Only in radioimmunoassays in which the MBP peptide 45-89 served as the radioligand could clearly elevated values of immunoreactive MBP be measured in CSF specimens from 5 patients with multiple sclerosis during or immediately after an exacerbation. The two antisera that reacted well with MBP peptide 80-89 resulted in higher levels of immunoreactive MBP measured in CSF. An epitope present in human MBP peptide 80-89 but sharing a conformation with both MBP and MBP peptide 45-89 is present in CSF following acute central nervous system myelin damage in multiple sclerosis.

Analysis of the Primary Sequence of Human Myelin Basic Protein Peptides 1–44 and 90–170 by Fast Atom Bombardment Mass Spectrometry

The originally described sequence of human myelin basic protein peptide 45-89 has recently been shown to contain two errors which have now been resolved. In the present study fast atom bombardment mass spectrometry was utilized to analyze the primary sequence of the other portions, peptides 1-44 and 90-170 of human myelin basic protein. The results obtained confirm the accuracy of the primary sequence published for both of these terminal peptides.

Multiple epitopes in a dodecapeptide of myelin basic protein determined by monoclonal antibodies.

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89.

Hormonal and drug effects on the degradation of human myelin basic protein peptide 43–88 by alkaline proteolytic activity in the rat kidney

The microsomal brush-border fraction of rat renal tissue contains enzymatic activity, optimally active at pH 9, that is capable of degrading human myelin basic protein (BP) peptide 43–88. In the present study, this degradation and the effect on it selected drugs and hormones were examined further. Of the substances tested, 10 −2M chloroquine and 10 −5M ACTH 1–24 were found to be the most effective inhibitors followed by 10 −5M ACTH 1–39; parathormone, glucagon and insulin were found to be inhibitors an order of magnitude weaker than ACTH 1–24. Hydrocortisone, dexamethasone, maleic acid and ACTH 4–10 were found to have minimal or no inhibitory effect on the peptide degrading activity. Gel filtration of the degradation products indicated that the rate of degradation of BP peptide 43–88 at pH 9 had been retarded by ACTH 1–24. These studies indicate that the clearance and catabolism of this peptide may be altered by available therapeutic agents.

Separation and isolation of human myelin basic protein peptides by reversed-phase high-performance liquid chromatography in volatile buffers

Separation and isolation of human myelin basic protein peptides by reversed-phase high-performance liquid chromatography in volatile buffers

Amino acid sequence of human myelin basic protein peptide 45-89 as determined by mass spectrometry.

In order to resolve the uncertainties about the primary structure of human myelin basic protein at residues 45-89, the sequence of this peptide and its tryptic fragments were reinvestigated by fast atom bombardment mass spectrometry. The sequence at positions 77-78 was found to be His-Gly and the sequence at positions 83-84 was shown to be Glu-Asn. The Ser at position 56 was not phosphorylated, whereas the residue at position 46 or 47 showed a heterogeneity of Gly and Ser in this peptide fragment in one of two protein preparations from different patients. These results demonstrate the usefulness of fast atom bombardment mass spectrometry for primary structure information. The corrected sequence of human basic protein peptide 45-89 will permit a more detailed immunochemical analysis of this peptide and its in vivo degradation products.

The degradation of human myelin basic protein peptide 43-88 by human renal neutral proteinase.

Normal human kidney contains a neutral endopeptidase that can degrade human myelin basic protein peptide 43-88. In the present study, renal homogenates prepared from postmortem tissue obtained from four persons with multiple sclerosis, two with amyotrophic lateral sclerosis, one each with olivopontocerebellar atrophy, subacute sclerosing panencephalitis, and Guillain-Barré syndrome, and four controls were analyzed for the enzymes present that degrade human myelin BP peptide 43-88. There was no evidence that the activity in renal tissue for degrading human BP peptide 43-88 is qualitatively different in persons with MS, other neurologic diseases, or controls. Gel filtration of digested peptide demonstrated the action of an endopeptidase capable of hydrolyzing BP peptide 43-88 into large fragments.

Species comparison of renal proteolytic activity for human myelin basic protein peptide 43–88

1. 1. A species comparison was conducted on the proteolytic activity in human, dog, rabbit, guinea-pig and rat kidney which can degrade human myelin basic protein peptide 43–88. 2. 2. In rat kidney the degrading activity occurred over a pH range of 4–11.5 with the greatest activities at pH 5 and 9. The peptide degrading activity in human, dog, rabbit and guinea-pig kidney was considerably less than in the rat and occurred predominantly at pH 7 with lesser activity at pH 9. 3. 3. The effects of inhibitors of proteolytic enzymes indicated that the peptide degrading activities at the same two pH's of dog, rabbit and guinea-pig were similar to one another but differed from that of human. 4. 4. These results indicate that the activity for degrading a potential autoantigenic material is widespread in renal tissue among different species and that different enzymes are involved. More generally, these findings suggest that renal proteinases differ among commonly used laboratory animals and also differ from the human enzymes.

The antigenic reactivity of small fragments derived from human myelin basic protein peptide 43-88.

In an effort to develop immunochemical reagents for detecting small peptides originating from myelin basic protein (BP), the antigenic determinants of fragments from human BP peptide 43-88 were examined. Antisera were produced in nine sheep and forty rabbits immunized with BP, BP peptide 43-88, or a region derived from within or containing a portion of BP peptide 43-88. These included custom synthesized peptides 51-67, 67-80, 74-84, 79-88, and 83-95. Reactivities were assessed by double antibody radioimmunoassay (DA-RIA) using radiolabeled BP or BP peptides. For peptides 74-84, 79-88, and 83-95 it was necessary to synthetically add a terminal tyrosine residue for radioiodination. Antisera to peptides 51-67, 67-80, 74-84, 79-88, and 83-95 showed much greater reactivity with the homologous antigen, with variable, but sometimes no, reactivity against BP or BP peptide 43-88. This was particularly evident in displacement DA-RIA. Of the small peptides, antisera to whole BP reacted best with peptide 83-95, whereas antisera to peptide 43-88 reacted best with peptide 79-88. Placement of the synthetically added tyrosine had a marked influence on the reactivity of BP peptide 79-88: antisera to BP peptide 43-88 reacted much better with radioiodinated tyrosinyl peptide 79-88 than with radioiodinated peptide 79-88-tyrosine. These results indicate that within a region of BP encompassing residues 51 through 95 a number of potential antigenic determinants exist. Some of the determinants on the small peptides represent distinctive conformational determinants or are inaccessible in BP peptide 43-88. The ability to detect small BP peptides by immunoassay necessitates that the identity of the peptide be known and that antibody reagents capable of reacting with the peptide(s) be available.