Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation.

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts (positions 1--51, 54--99, and 102--127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

Early pregnancy factor suppresses experimental autoimmune encephalomyelitis induced in Lewis rats with myelin basic protein and in SJL/J mice with myelin proteolipid protein peptide 139-151.

Early pregnancy factor (EPF) is a secreted protein with immunosuppressive and growth factor properties. During pregnancy, it appears in maternal serum within 6-24 h of fertilization, is present for at least the first two-thirds of pregnancy in all species studied and is essential for embryonic survival. It is a homologue of chaperonin 10, a heat shock protein, but, unlike other members of this family, EPF has an extracellular role. As it has the ability to modulate CD4+ T cell-dependent immune responses, its role in treatment of experimental autoimmune encephalomyelitis (EAE) was investigated. EAE is a CD4+ T cell-mediated disease, the best available animal model of multiple sclerosis (MS). Two models of EAE were investigated, acute EAE induced in Lewis rats by inoculation with myelin basic protein (MBP-EAE) and chronic relapsing EAE induced in SJL/J mice by inoculation with myelin proteolipid protein peptide (residues 139-151) (PLP-EAE). EPF, delivered intraperitoneally or orally to rats or intraperitoneally to mice, suppressed clinical signs of disease. Mice with PLP-EAE were also treated with interferon-beta, with and without EPF. Both EPF and IFN-beta suppressed clinical signs of EAE and, when administered together, gave greater suppression than when given separately. These findings suggest that EPF may be a potential candidate for use in treatment of MS and may be of use in combined therapy with IFN-beta.

Synthetic, structural and biological studies of the ubiquitin system: synthesis and crystal structure of an analogue containing unnatural amino acids.

Ubiquitin is a 76-amino acid protein involved in the targeting for destruction of proteins in the cell. The protein can readily be synthesized chemically affording an extra dimension to studies of protein stability. Ubiquitin with various modifications to the hydrophobic core has been synthesized. In particular, two core amino acids have been replaced by aminobutyric acid (Val-26) and norvaline (for Ile-30) and the product crystallized. The refined crystal structure shows an overall contraction of the molecule and the side chain of Nva-30 rotates relative to Ile-30. However, the side chain rotation is not sufficient to compensate for the effect of the loss of the methyl group and hence a small cavity is introduced into the structure, which decreases the stability of the protein. The biological behaviour of the modified protein is unaltered. The observed changes in stability are of the magnitude expected for the removal of methyl groups from the hydrophobic core of a protein. Interestingly, the effect appears to be independent of the position of the removed methyl group. The intact structure, but not its stability, is important for recognition by the biological conjugating system.

Origin of asteroid rotation rates in catastrophic impacts.

The rotation rates of asteroids, which are deduced from periodic fluctuations in their brightnesses, are controlled by mutual collisions. The link between asteroid spin and collision history is usually made with reference to impact experiments on centimetre-scale targets, where material strength governs the impact response. Recent work, however, indicates that for objects of the size of most observed asteroids (> or = 1 km in diameter), gravity rather than intrinsic strength controls the dynamic response to collisions. Here we explore this idea by modelling the effect of impacts on large gravitating bodies. We find that the fraction of a projectile's angular momentum that is retained by a target asteroid is both lower and more variable than expected from laboratory experiments, with spin evolution being dominated by 'catastrophic' collisions that eject approximately 50 per cent of the target's mass. The remnant of an initially non-rotating silicate asteroid that suffers such a collision rotates at a rate of approximately 2.9 per day, which is close to the observed mean asteroid rotation rate of approximately 2.5 d-1. Moreover, our calculations suggest that the observed trend in the mean spin frequency for different classes of asteroids (2.2 d-1 for C-type asteroids, 2.5 d-1 for S-type, and 4.0 d-1 for M-type) is due to increasing mean density, rather than increasing material strength.

Reversal of T cell unresponsiveness by augmentation of antigen presenting cell function.

We have previously described epitopes of the 18 kDa protein of Mycobacterium leprae which stimulate T and B cell responses in different strains of mice. A series of overlapping 20-mer peptides that span the 18 kDa protein were used as immunogens to examine T and B cell recognition of different epitopes. Strain-specific variation in the epitopes which induce the strongest responses was affected by genes linked to the H-2 complex and the T cell responses revealed by re-challenge with antigen were at least partially controlled by factors other than T cell specificity. We have examined the responses to one such antigen, peptide 1-20, which contains strongly immunogenic epitopes for T and B cells. T cells from draining lymph nodes of peptide 1-20 immunized B10.BR, but not BALB/c mice, proliferated in vitro in response to rechallenge with peptide 1-20 or whole protein. Immunization with the same peptide also induced specific antibody only in B10.BR mice. However, immunization of BALB/c mice results in 'silent' priming of T cells since these can be induced to respond in vitro to this antigen when cultured with activated macrophages as antigen presenting cells (APC). The failure of APC from mBALB/c mice primed with peptide 1-20 to stimulate CD4+ proliferation when re-challenged in vitro and the failure to elicit antibody responses to peptide 1-20 are presumably due to the same defect in antigen-presenting cell function, since presentation of peptide 1-20 by activated macrophages is sufficient to restore both responses.(ABSTRACT TRUNCATED AT 250 WORDS)

A direct measurement of the terrestrial mass accretion rate of cosmic dust.

The mass of extraterrestrial material accreted by the Earth as submillimeter particles has not previously been measured with a single direct and precise technique that samples the particle sizes representing most of that mass. The flux of meteoroids in the mass range 10(-9) to 10(-4) grams has now been determined from an examination of hypervelocity impact craters on the space-facing end of the Long Duration Exposure Facility satellite. The meteoroid mass distribution peaks near 1.5 x 10(-5) grams (200 micrometers in diameter), and the small particle mass accretion rate is (40 +/- 20) x 106 kilograms per year, higher than previous estimates but in good agreement with total terrestrial mass accretion rates found by geochemical methods. This mass input is comparable with or greater than the average contribution from extraterrestrial bodies in the 1-centimeter to 10-kilometer size range.

Identification of T cell stimulatory epitopes from the 18 kDa protein of Mycobacterium leprae.

We have used different mouse strains to examine in vivo and in vitro responses to the 18 kDa protein of Mycobacterium leprae, which appears to be strongly immunogenic in both mice and humans. B and T cell stimulatory epitopes recognised by different strains of mice have been mapped using overlapping peptides that span the entire 18 kDa protein. Previous work established that immunization of mice with the 18 kDa protein results in specific antibody production to common B cell epitopes and immunization of mice with peptides containing these B cell epitopes resulted in the induction of specific IgG to only a limited subset of epitopes in each strain. Now we report that T cells purified from mice immunized with peptides that stimulate antibody production, proliferate in vitro when rechallenged. The proliferating T cells produce levels of IL-2 and IFN-gamma, that indicate antigen-specific T helper type 1 cells are present in significant numbers. Thus, a comparison of in vivo and in vitro data suggests that T cells bearing the phenotype associated with potentially protective cell-mediated responses can be primed in vivo by epitopes on small peptides. Since T cells from both strains of mice are capable of responding to the immunogenic synthetic peptides in vitro, but give different responses to the same peptides in vivo, factors other than epitope structure appear to influence T cell subset activation. This may have important implications for diseases such as leprosy where a polarized T cell response appears to develop and for the development of synthetic subunit vaccines.

Immune responses to the 18-kDa protein of Mycobacterium leprae. Similar B cell epitopes but different T cell epitopes seen by inbred strains of mice.

Antibody responses to the 18-kDa protein of Mycobacterium leprae have been analyzed in different strains of mice. High, intermediate, and low responder strains have been identified and these response patterns show clear linkage to genes encoded in the H-2 complex. Three peptides, residues 1-50, 51-100, and 101-148 have been synthesized, as well as a series of 20-mer peptides, which span the entire 18-kDa protein. Repeated immunization of different strains of mice with the 18-kDa protein resulted in IgG responses to epitopes found on all three synthetic peptides. Immunization of BALB/cJ and B10.BR mice, two high responder strains, with 18-kDa protein resulted in high levels of IgG antibody to epitopes found on peptides 1-20, 16-35, 31-50, 46-65, and 76-95. B10.BR mice also contained IgG that bound peptide 61-80 and BALB/cJ mice produced IgG that bound peptide 91-110. Although B10.BR mice produced IgG that bound the 50-mer peptide 101-148, this IgG was not detected by binding to peptides 91-110, 106-125, 121-140, and 131-148. Immunization of B10.BR mice with individual overlapping 20-mer peptides as Ag revealed that peptides 1-20, 16-35, 31-50, and 76-95 elicited high titers of IgG that bound both the immunizing peptide as well as 18-kDa protein. As these peptides induce antibody synthesis they must contain both B cell and T cell epitopes. By contrast, immunization of BALB/cJ mice with the same 20-mer peptides, all of which contain B cell epitopes for this strain, failed to elicit IgG responses with one exception. Peptide 91-110 induced IgG that bound peptide 91-110, but not the intact 18-kDa protein. We conclude that peptides 1-20, 16-35, 31-50, and 76-95 either lack T cell epitopes for BALB/cJ mice, or activate different T cell subpopulations in the two strains. We suggest that the induction of IgG responses to small peptide Ag is an in vivo assay of the activity of Th2 cell subpopulations.

The mapping of epitopes of the 18-kDa protein of Mycobacterium leprae recognized by murine T cells in a proliferation assay.

The 18-kDa protein of Mycobacterium leprae was purified from recombinant plasmids pUL108 and pML-3 grown in Saccharomyces cerevisiae and Escherichia coli, respectively. Significant lymphoproliferative responses were observed when T cells from immunized mice were challenged in culture with purified 18-kDa protein. Synthetic peptides have been prepared that span most of the 148 amino acid residues that constitute the sequence of the 18-kDa protein and used to map epitopes recognized by T cells. When mice were immunized with 18-kDa protein and lymph node cells subsequently prepared and challenged in microculture proliferative assays by using synthetic peptides, only one region of the intact protein appeared stimulatory. This T cell epitope was located between residues 116 and 121, adjacent to an epitope between residues 110 and 115 which we have previously shown to bind the L5 mAb. Immunization of mice with peptides, and subsequent challenge of lymph node cells in assays by using the 18-kDa protein as Ag revealed that residues 111-125 were the most effective in priming responses. Furthermore, the ability of 18-kDa primed lymph node cells to recognize determinants on both M. leprae and Mycobacterium tuberculosis indicates that in addition to possessing an M. leprae-specific B cell determinant, the 18-kDa protein contains a cross-reactive T cell epitope(s).

Characterization of an antibody-binding epitope from the 18-kDa protein on Mycobacterium leprae.

A murine mAb, designated L5, appears to be specific for an epitope on a protein from Mycobacterium leprae of restricted distribution within the mycobacteria. This protein, of Mr 18,000 (18 kDa) is of interest because monoclonal antibodies raised against it do not appear to cross-react with other mycobacterial pathogens. The L5 antibody-binding epitope has been mapped by two complementary methods; expression of gene fragments and synthesis of short peptides. This L5-binding region of the 18-kDa protein (amino acids 109 to 115) shows some homology to a region of the GroEL heat shock family of proteins. Characterization of this antibody-binding epitope may lead to a reagent of use in early diagnosis of infection.