Gbetagamma affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the gamma subunit.

Two native betagamma dimers, beta(1)gamma(1) and beta(1)gamma(2), display very different affinities for receptors. Since these gamma subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gbetagamma interaction with receptors. We constructed baculoviruses encoding gamma(1) and gamma(2) subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the gamma chains and a set of gamma(1) and gamma(2) chimeras with the gamma(2) CAAX motif at the carboxyl terminus. All the gamma constructs coexpressed with beta(1) in Sf9 cells yielded beta(1)gamma dimers, which were purified to near homogeneity, and their affinities for receptors and Galpha were quantitatively determined. Whereas alteration of the isoprenoid of gamma(1) from farnesyl to geranylgeranyl and of gamma(2) from geranylgeranyl to farnesyl had no impact on the affinities of beta(1)gamma dimers for Galpha(t), the non-prenylated beta(1)gamma(2) dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the beta(1)gamma(2) dimer for rhodopsin and a <3-fold change for the beta(1)gamma(1) dimer. In each case with identical isoprenylation, the beta(1)gamma(2) dimer displayed significantly greater affinity for rhodopsin compared with the beta(1)gamma(1) dimer. Furthermore, dimers containing chimeric Ggamma chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Ggamma subunit is essential for binding to both Galpha and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Galpha. Finally, the primary structure of the Ggamma subunit provides a major contribution to receptor binding of Gbetagamma, with the carboxyl-terminal sequence conferring receptor selectivity.

Three distinct stages of lens opacification in transgenic mice expressing the HIV-1 protease.

Scanning electron microscopy of the lenses from transgenic mice (TG(72)) containing the HIV-1 protease linked to the lens alphaA-crystallin promoter showed structural changes around postnatal day 16. Frank opacification of the lens was observed at day 24. To relate the biochemical and biophysical changes that occur during the process of cataract development, high-resolution two-dimensional gel electrophoresis (2D), quantitative image analysis and ion measurements were carried out on lenses from postnatal day 10 and on days 15-24. The phase separation temperature (Tc), a measure of molecular interactions between proteins, was also determined for normal and transgenic lenses. A comparison of the transgenic and normal lenses on day 10 revealed no significant differences in any of the measured parameters. However, starting around day 16 or the first stage of observed structural changes, the TG(72)crystallin profiles of the alphaA- alphaB-, betaA3-, betaA4-, betaB3 and one gamma-crystallin began to deviate from the normal. By postnatal day 20, a second stage was initiated with an influx of calcium and sodium ions that was accompanied by modifications of betaB1- and betaB2-crystallin. In the third and final stage of the cataract process, a large increase in the proteolysis of crystallins was accompanied by the appearance of the frank cataract on day 24. The Tc initially increased in all of the mouse lenses until just prior to eyelid opening. After that time, the Tc decreased in all lenses. Although the Tc continued to decrease in the normal lenses with age, for the homozygous transgenic mice it exhibited a dramatic increase that began on day 20. Thus, in the TG(72)transgenic mouse, cataract formation occurs in a three-stage process. Tc and other biophysical parameters previously measured appeared to be insensitive to the modifications that occur during stage 1. However, during the second stage of cataract formation, there was a correspondence between abnormal Tc and the abnormal interactions between cellular constituents apparently resulting from lens hydration, the loss of ion homeostasis and continued proteolysis. The last stage of cataract formation results in a total loss of lens transparency and leakage of lens proteins.

Complement factor H is a serum-binding protein for adrenomedullin, and the resulting complex modulates the bioactivities of both partners.

Adrenomedullin (AM) is an important regulatory peptide involved in both physiological and pathological states. We have previously demonstrated the existence of a specific AM-binding protein (AMBP-1) in human plasma. In the present study, we developed a nonradioactive ligand blotting assay, which, together with high pressure liquid chromatography/SDS-polyacrylamide gel electrophoresis purification techniques, allowed us to isolate AMBP-1 to homogeneity. The purified protein was identified as human complement factor H. We show that AM/factor H interaction interferes with the established methodology for quantification of circulating AM. Our data suggest that this routine procedure does not take into account the AM bound to its binding protein. In addition, we show that factor H affects AM in vitro functions. It enhances AM-mediated induction of cAMP in fibroblasts, augments the AM-mediated growth of a cancer cell line, and suppresses the bactericidal capability of AM on Escherichia coli. Reciprocally, AM influences the complement regulatory function of factor H by enhancing the cleavage of C3b via factor I. In summary, we report on a potentially new regulatory mechanism of AM biology, the influence of factor H on radioimmunoassay quantification of AM, and the possible involvement of AM as a regulator of the complement cascade.

Tracking pathology with proteomics: identification of in vivo degradation products of alphaB-crystallin.

Soemmerring's ring is one form of "after cataract" that can occur following cataract surgery. The ring structure is formed by adherence of the anterior lens capsule to the posterior lens capsule. Epithelial cells remaining after surgery differentiate into lens fiber cells but the resulting tissue mass does not remain transparent. The protein in normal lens and in Soemmerring's rings from four individuals was analyzed using two-dimensional (2-D) gel electrophoresis, matrix assisted laser desorption/ionization-time of-flight-mass spectrometry (MALDI-TOF-MS) and image analysis with Phoretix software. The 2-D protein patterns of the Soemmerring's rings were generally similar to that of cortical fiber cells of normal human lens with some notable exceptions. Several post-translationally modified forms of alphaB-crystallin(1-175) were identified. Two degradation products, alphaB-crystallin(1-170) and alphaB-crystallin(1-174), each make up 9.5-27% of the total alphaB-crystallin in the Soemmerring's rings and less than 1% in the normal lenses. Other modified forms of alphaB-crystallin are aberrant in the fiber cells of the Soemmerring rings relative to normal lens.

Identities and phylogenetic comparisons of posttranscriptional modifications in 16 S ribosomal RNA from Haloferax volcanii.

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T(1), accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N(6)-methyladenosine-1501, and N(6),N(6)-dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N(6)-methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

An atypical form of alphaB-crystallin is present in high concentration in some human cataractous lenses. Identification and characterization of aberrant N- and C-terminal processing.

Two unique polypeptides, 22.4 and 16.4 kDa, were prominent in some human cataracts. Both proteins were identified as modified forms of the small heat shock protein, alphaB-crystallin. The concentration of total alphaB-crystallin in most of these cataracts was significantly increased. The 22.4-kDa protein was subsequently designated as alphaB(g). Mass spectrometric analyses of tryptic and Asp-N digests showed alphaB(g) is alphaB-crystallin minus the C-terminal lysine. alphaB(g) constituted 10-90% of the total alphaB-crystallin in these cataracts and was preferentially phosphorylated over the typical form of alphaB-crystallin. Human alphaB(g) and alphaB-crystallin were cloned and expressed in Escherichia coli. The differences in electrophoretic mobility and the large difference in native pI values suggest some structural differences exist. The chaperone-like activity of recombinant human alphaB(g) was comparable to that of recombinant human alphaB-crystallin in preventing the aggregation of lactalbumin induced by dithiothreitol. The mechanism involved in generating alphaB(g) is not known, but a premature termination of the alphaB-crystallin gene was ruled out by sequencing the polymerase chain reaction products of the last exon for the alphaB-crystallin gene from lenses containing alphaB(g). The 16.4-kDa protein was an N-terminally truncated fragment of alphaB(g). The high concentration of alphaB-crystallin in these cataracts is the first observation of this kind in human lenses.

Studies towards neoglycoconjugates from the monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa using the diethyl squarate reagent.

The effect of reaction time, concentration and molar excess of hapten upon the efficiency of the conjugation of carbohydrates to proteins using the diethyl squarate reagent has been studied using chicken serum albumin (CSA) as the carrier protein and a linker-equipped D-glucose derivative as the hapten. A high degree of incorporation of the latter into CSA was achieved with high efficiency, and the use of a large excess of the ligand was not necessary. Conjugation of the immunodominant monosaccharide determinant of Vibrio cholerae O:1, serotype Ogawa, bearing the same spacer, followed a similar pattern, showing that the nature of the carbohydrate does not substantially affect the outcome of the conjugation and that a predicted degree of antigen-loading onto carrier protein is possible to achieve.

Beta-methylthio-aspartic acid: identification of a novel posttranslational modification in ribosomal protein S12 from Escherichia coli.

Utilizing microscale chemical derivatization reactions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we have identified a novel posttranslational modification of aspartic acid, beta-methylthio-aspartic acid. The modified residue is located at position 88 in ribosomal protein S12 from Escherichia coli, a phylogenetically conserved protein that has been implicated in maintaining translational accuracy of the ribosome.

Primary structure of a new neuropeptide, cerebral peptide 2, purified from cerebral ganglia of Aplysia.

We report the purification and characterization of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed cerebral peptide 2 (CP2) because it was the larger of two peptides predominantly synthesized in the cerebral ganglia and transported to other regions of the central nervous system. The purification of CP2 from extracts of cerebral ganglia using three sequential modes of high-pressure liquid chromatography (HPLC) was followed using the [35S]methionine-labeled peptide obtained from transport experiments. The primary structure of CP2 was determined by automated Edman degradation of native CP2 and its proteolytic fragments in conjunction with mass spectrometry. CP2 is a 41 amino acid peptide with an amidated carboxyl terminal. A peptide with the proposed sequence of CP2 was synthetized and compared by HPLC with the native peptide. Chromatographic properties of the synthetic and native peptide labeled in vivo were found to be identical. CP2 does not appear to be a member of any previously identified peptide family.

Structural characterization of U*-1915 in domain IV from Escherichia coli 23S ribosomal RNA as 3-methylpseudouridine.

Mass spectrometry-based methods have been used to study post-transcriptional modification in the 1900-1974 nt segment of domain IV in 23S rRNA of Escherichia coli, a region which interacts with domain V in forming the three- dimensional structure of the peptidyl transferase center within the ribosome. A nucleoside constituent of M r 258 (U*)which occurs at position 1915, within the highly modified oligonucleotide sequence 1911-psiAACU*Apsi-1917, was characterized as 3-methylpseudouridine (m3psi). The assignment was confirmed by chemical synthesis of m3psi and comparison with the natural nucleoside by liquid chromatography-mass spectrometry. 3-Methylpseudouridine is previously unknown in nature and is the only known derivative of the common modified nucleoside pseudouridine thus far found in bacterial rRNA.

Posttranscriptional modification of the central loop of domain V in Escherichia coli 23 S ribosomal RNA.

Knowledge of the sites, structures, and functional roles of posttranscriptional modification in rRNAs is limited, despite steadily accumulating evidence that rRNA plays a direct role in the peptidyl transferase reaction and that modified nucleotides are concentrated at the functional center of the ribosome. Using methods based on mass spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the central loop of domain V, a region of established interaction between 23 S RNA and tRNA. Two segments of RNA were isolated following protection with oligodeoxynucleotides and nuclease digestion: residues 2423-2473 (51-mer) and 2481-2519 (39-mer). Dihydrouridine was located at position 2449, within the RNase T1 hydrolysis product 2448-ADAACAGp-2454, as evidenced by a molecular mass 2 daltons higher than the gene sequence-predicted mass. This nucleoside, which is nearly ubiquitous in tRNA (where it is involved in maintenance of loop structure), is two bases from A-2551, a previously determined site of interaction between 23 S RNA and the CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately mass measured, and its nucleoside constituents were characterized by high performance liquid chromatography-mass spectrometry; there was no evidence of modification at position 2501 as implied by earlier work. Using similar techniques, the modified adenosine at position 2503 was unambiguously determined to be 2-methyladenosine in the fragment 2503-m2A psi Gp-2505.

The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516.

The number and location of pseudouridine residues in Escherichia coli 16S ribosomal RNA has been determined by a combination of direct and indirect methods. Only one residue was found, at position 516. This site is at the 5'-end of one of the three most highly conserved long sequences of this RNA molecule. A number of experimental findings have strongly implicated this loop in the fidelity of codon recognition by A-site bound tRNA. By virtue of its location, we suggest that psi 516 may also play a role in maintaining the fidelity of protein synthesis.

The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles.

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.

beta-D-glucosyl-hydroxymethyluracil: a novel modified base present in the DNA of the parasitic protozoan T. brucei.

We have previously shown that the DNA of the unicellular eukaryote T. brucei contains about 0.1% of a novel modified base, called J. The presence of J correlates with a DNA modification associated with the silencing of telomeric expression sites for the variant surface antigens of trypanosomes. Here we show that J is 5-((beta-D-glucopyranosyloxy)-methyl)-uracil (shortened to beta-D-glucosyl-hydroxymethyluracil), a base not previously found in DNA. We discuss putative pathways for the introduction of this base modification at specific positions in the DNA and the possible contribution of this modification to repression of surface antigen gene expression.

A novel method for the determination of post-transcriptional modification in RNA by mass spectrometry.

A method is described for the detection, chemical characterization and sequence placement of post-transcriptionally modified nucleotides in RNA. Molecular masses of oligonucleotides produced by RNase T1 hydrolysis can be measured by electrospray mass spectrometry with errors of less than 1 Da, which provides exact base composition, and recognition of modifications resulting from incremental increases in mass. Used in conjunction with combined liquid chromatography-mass spectrometry and gene sequence data, modified residues can be completely characterized at the nucleoside level, and assigned to sequence sites within oligonucleotides defined by selective RNase cleavage. The procedures are demonstrated using E.coli 5S rRNA, in which all RNase T1 fragments predicted from the rDNA sequence are identified solely on the basis of their molecular masses, and using E.coli 16S rRNA for analysis of post-transcriptional modification, including placement of 3-methyluridine at position 1498. The principles described are generally applicable to other covalent structural modifications of RNA which produce a change in mass, such as those resulting from editing, photochemical cross-linking, or xenobiotic modification.

5S rRNA modification in the hyperthermophilic archaea Sulfolobus solfataricus and Pyrodictium occultum.

The 5S rRNAs from Sulfolobus solfataricus and Pyrodictium occultum were digested to nucleosides and analyzed using directly-combined HPLC/mass spectrometry. P. occultum 5S rRNA contains two modified nucleoside species, N4-acetylcytidine (ac4C) and N4-acetyl-2'-O-methylcytidine (ac4Cm). Oligonucleotides were generated from P. occultum 5S rRNA by RNase T1 hydrolysis, and their molecular weights were determined using electrospray mass spectrometry and compared with those predicted from the P. occultum 5S RNA gene sequence. Deviation in mass between expected and observed molecular weights permitted ac4Cm to be located at position 35, in the nonanucleotide CAA-CACC[ac4Cm]G, and the ac4C in one or both of two (C,U)G trinucleotides. 2'-O-Methylcytidine is unambiguously characterized in S. solfataricus 5S rRNA, confirming earlier tentative assignments at the analogous sequence position (Stahl, D.A., Luehrsen, K.R., Woese, C.R., and Pace, N.R. (1981) Nucleic Acids Res., Vol. 9, pp. 6129-6137; Dams, E., Londei, P., Cammarano, P., Vandenberghe, A., and De Wachter, R. (1983) Nucleic Acids Res. Vol. 11, pp. 4667-4676). Potential effects of the presence of ac4C and ac4Cm on thermal stabilization of 5S rRNA in thermophiles are discussed.

Determination of oligonucleotide composition from mass spectrometrically measured molecular weight.

Extensive calculations for molecular mass versus subunit composition have been made for oligonucleotides from RNA and DNA to determine the extent to which base compositions might be derived from mass spectrometrically determined molecular weights. In the absence of compositional constraints (e.g., any numbers of A, U, G, C), measurement of molecular weight leads to only modest restrictions in allowable number of base compositions; however, if the compositional value for any one residue is known, such as from selective chemical modification or enzymatic cleavage, the number of allowable base compositions becomes unexpectedly low. For example, hydrolysis of RNA by ribonuclease T1 produces oligonucleotides for which G=1, for which all base compositions can be uniquely specified up to the 14-mer level, solely by measurement of mass to within ±0,01%. The effects of methylation, phosphorylation state of nucleotide termini, and knowledge of chain length on the determination of subunit composition are discussed.

Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria).

Nucleoside modification has been studied in unfractionated tRNA from 11 thermophilic archaea (archaebacteria), including phylogenetically diverse representatives of thermophilic methanogens and sulfur-metabolizing hyperthermophiles which grow optimally in the temperature range of 56 (Thermoplasma acidophilum) to 105 degrees C (Pyrodictium occultum), and for comparison from the most thermophilic bacterium (eubacterium) known, Thermotoga maritima (80 degrees C). Nine nucleosides are found to be unique to the archaea, six of which are structurally novel in being modified both in the base and by methylation in ribose and occur primarily in tRNA from the extreme thermophiles in the Crenarchaeota of the archaeal phylogenetic tree. 2-Thiothymine occurs in tRNA from Thermococcus sp., and constitutes the only known occurrence of the thymine moiety in archaeal RNA, in contrast to its near-ubiquitous presence in tRNA from bacteria and eukarya. A total of 33 modified nucleosides are rigorously characterized in archaeal tRNA in the present study, demonstrating that the structural range of posttranscriptional modifications in archaeal tRNA is more extensive than previously known. From a phylogenetic standpoint, certain tRNA modifications occur in the archaea which are otherwise unique to either the bacterial or eukaryal domain, although the overall patterns of modification are more typical of eukaryotes than bacteria.