Astrochemistry: Complex organic matter in Titan's aerosols?

On 14 January 2005, the Huygens probe entered the atmosphere of Titan after a seven-year interplanetary flight as part of the Cassini mission to Saturn. Huygens carried, among other instruments, an aerosol collection and pyrolysis (ACP) device. Its designers, Israël et al., now claim to have detected complex organic matter in two aerosol samples collected at different altitudes (130-35 km and 25-20 km, respectively), on the basis of their detection of ammonia (NH3) and hydrogen cyanide (HCN) when the sample oven was heated to 600 degrees C. However, the authors' remarkable conclusions, which would have far-reaching consequences for our understanding of the chemical environment prevailing on Saturn's largest moon, are not supported by their limited data.

Astrochemistry: Complex organic matter in Titan's aerosols?

On 14 January 2005, the Huygens probe entered the atmosphere of Titan after a seven-year interplanetary flight as part of the Cassini mission to Saturn. Huygens carried, among other instruments, an aerosol collection and pyrolysis (ACP) device. Its designers, Israël et al., now claim to have detected complex organic matter in two aerosol samples collected at different altitudes (130-35 km and 25-20 km, respectively), on the basis of their detection of ammonia (NH3) and hydrogen cyanide (HCN) when the sample oven was heated to 600 degrees C. However, the authors' remarkable conclusions, which would have far-reaching consequences for our understanding of the chemical environment prevailing on Saturn's largest moon, are not supported by their limited data.

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site.

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is DeltaU(2S)H(NS,6S)I(2S)H(NS, 6S)I(2S)H(NS,6S)IH(NAc,6S)GH(NS,3S,6S) (+/-DDD4-7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin.

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Determination of the carbohydrate composition and the disulfide bond linkages of bovine lactoperoxidase by mass spectrometry.

The extent and distribution of N-glycosylation and the nature of most of the disulfide bond linkages were determined for bovine lactoperoxidase through proteolytic and glycolytic digestions combined with matrix-assisted laser desorption/ionization mass spectrometric analysis. In addition, 98% of the primary sequence of the protein was confirmed. All five of the asparagines present in sequons were found to be glycosylated, predominantly by high mannose and complex structures. Six disulfide bonds were assigned, including Cys 32-Cys 45, Cys 146-Cys 156, Cys 150-Cys 174, Cys 254-Cys 265, Cys 473-Cys 530 and Cys 571-Cys 596.

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells.

BACKGROUND: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESULTS: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.

Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).

Mass spectrometric evidence for the enzymatic mechanism of the depolymerization of heparin-like glycosaminoglycans by heparinase II.

Heparin-like glycosaminoglycans, acidic complex polysaccharides present on cell surfaces and in the extracellular matrix, regulate important physiological processes such as anticoagulation and angiogenesis. Heparin-like glycosaminoglycan degrading enzymes or heparinases are powerful tools that have enabled the elucidation of important biological properties of heparin-like glycosaminoglycans in vitro and in vivo. With an overall goal of developing an approach to sequence heparin-like glycosaminoglycans using the heparinases, we recently have elaborated a mass spectrometry methodology to elucidate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase I. In this study, we investigate the mechanism of depolymerization of heparin-like glycosaminoglycans by heparinase II, which possesses the broadest known substrate specificity of the heparinases. We show here that heparinase II cleaves heparin-like glycosaminoglycans endolytically in a nonrandom manner. In addition, we show that heparinase II has two distinct active sites and provide evidence that one of the active sites is heparinase I-like, cleaving at hexosamine-sulfated iduronate linkages, whereas the other is presumably heparinase III-like, cleaving at hexosamine-glucuronate linkages. Elucidation of the mechanism of depolymerization of heparin-like glycosaminoglycans by the heparinases and mutant heparinases could pave the way to the development of much needed methods to sequence heparin-like glycosaminoglycans.

Direct evidence for a predominantly exolytic processive mechanism for depolymerization of heparin-like glycosaminoglycans by heparinase I.

Heparinase I from Flavobacterium heparinum has important uses for elucidating the complex sequence heterogeneity of heparin-like glycosaminoglycans (HLGAGs). Understanding the biological function of HLGAGs has been impaired by the limited methods for analysis of pure or mixed oligosaccharide fragments. Here, we use methodologies involving MS and capillary electrophoresis to investigate the sequence of events during heparinase I depolymerization of HLGAGs. In an initial step, heparinase I preferentially cleaves exolytically at the nonreducing terminal linkage of the HLGAG chain, although it also cleaves internal linkages at a detectable rate. In a second step, heparinase I has a strong preference for cleaving the same substrate molecule processively, i.e., to cleave the next site toward the reducing end of the HLGAG chain. Computer simulation showed that the experimental results presented here from analysis of oligosaccharide degradation were consistent with literature data for degradation of polymeric HLGAG by heparinase I. This study presents direct evidence for a predominantly exolytic and processive mechanism of depolymerization of HLGAG by heparinase I.

Mass spectrometric and capillary electrophoretic investigation of the enzymatic degradation of heparin-like glycosaminoglycans.

Difficulties in determining composition and sequence of glycosaminoglycans, such as those related to heparin, have limited the investigation of these biologically important molecules. Here, we report methodology, based on matrix-assisted laser desorption ionization MS and capillary electrophoresis, to follow the time course of the enzymatic degradation of heparin-like glycosaminoglycans through the intermediate stages to the end products. MS allows the determination of the molecular weights of the sulfated carbohydrate intermediates and their approximate relative abundances at different time points of the experiment. Capillary electrophoresis subsequently is used to follow more accurately the abundance of the components and also to measure sulfated disaccharides for which MS is not well applicable. For those substrates that produce identical or isomeric intermediates, the reducing end of the carbohydrate chain was converted to the semicarbazone. This conversion increases the molecular weight of all products retaining the reducing terminus by the "mass tag" (in this case 56 Da) and thus distinguishes them from other products. A few picomoles of heparin-derived, sulfated hexa- to decasaccharides of known structure were subjected to heparinase I digestion and analyzed. The results indicate that the enzyme acts primarily exolytically and in a processive mode. The methodology described should be equally useful for other enzymes, including those modified by site-directed mutagenesis, and may lead to the development of an approach to the sequencing of complex glycosaminoglycans.

Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin.

BACKGROUND: Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown. RESULTS: Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities. CONCLUSIONS: The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Identification of N-linked glycosylation sites in human testis angiotensin-converting enzyme and expression of an active deglycosylated form.

The sites of glycosylation of Chinese hamster ovary cell expressed testicular angiotensin-converting enzyme (tACE) have been determined by matrix-assisted laser desorption ionization/time of flight/mass spectrometry of peptides generated by proteolytic and cyanogen bromide digestion. Two of the seven potential N-linked glycosylation sites, Asn90 and Asn109, were found to be fully glycosylated by analysis of peptides before and after treatment with a series of glycosidases and with endoproteinase Asp-N. The mass spectra of the glycopeptides exhibit characteristic clusters of peaks which indicate the N-linked glycans in tACE to be mostly of the biantennary, fucosylated complex type. This structural information was used to demonstrate that three other sites, Asn155, Asn337, and Asn586, are partially glycosylated, whereas Asn72 appears to be fully glycosylated. The only potential site that was not modified is Asn620. Sequence analysis of tryptic peptides obtained from somatic ACE (human kidney) identified six glycosylated and one unglycosylated Asn. Only one of these glycosylation sites had a counterpart in tACE. Comparison of the two proteins reveals a pattern in which amino-terminal N-linked sites are preferred. The functional significance of glycosylation was examined with a tACE mutant lacking the O-glycan-rich first amino-terminal 36 residues and truncated at Ser625. When expressed in the presence of the alpha-glucosidase I inhibitor N-butyldeoxynojirimycin and treated with endoglycosidase H to remove all but the terminal N-acetylglucosamine residues, it retained full enzymatic activity, was electrophoretically homogeneous, and is a good candidate for crystallographic studies.

Assignment of the disulfide bonds in napin, a seed storage protein from Brassica napus, using matrix-assisted laser desorption ionization mass spectrometry.

The sites of the disulfide bonds in a napin protein isolated from Brassica napus have been identified by proteolytic cleavage and subsequent peptide mapping by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Napins consist of two polypeptide chains containing two and six cysteine residues, respectively, that are held together by disulfide bonds. Upon initial cleavage of native napin by Endo-Lys-C, a disulfide-linked core complex of four peptides was obtained. This core peptide was isolated by reversed-phase HPLC and further digested by thermolysin, and the resulting fragments were identified by MALDI-MS. In a separate set of experiments, intact napin was subjected to proteolysis by thermolysin, and an isolated disulfide-linked peptide of interest was subdigested again using thermolysin. The combined data resulting from these experiments allowed the assignment of the disulfide linkages in a relatively abundant napin isoform, BngNAP1, apart from an ambiguity concerning the adjacent cysteines at positions 14' and 15' of the long chain. Two intermolecular disulfide bonds link Cys10 (short chain) with Cys25' (long chain) and Cys23 with Cys14' (or Cys15'), respectively. The long chain of napin contains two intramolecular disulfide bonds connecting Cys27' with Cys80' and Cys14' (or Cys15') with Cys72'.

Identification of an active site residue of the R1 subunit of ribonucleotide reductase from Escherichia coli: characterization of substrate-induced polypeptide cleavage by C225SR1.

Incubation of the C225S mutant of the R1 subunit of ribonucleotide reductase from Escherichia coli with the R2 subunit and nucleoside diphosphates leads to fragmentation of the polypeptide backbone of R1 [Mao, S. S., Holler, T. P., Bollinger, J.M., Jr., Yu, G. X., Johnston, M.I., & Stubbe, J. (1992) Biochemistry 31, 9744--9751]. The 26 and 60 kDa cleavage fragments were purified to homogeneity. The 26 kDa polypeptide was digested with Lys-C, and the peptides were partially purified by RP-HPLC. Mass spectrometric analysis (MALDI-TOF) of the HPLC fractions allowed the identification of the C-terminal peptide. The molecular mass of this peptide (2176) revealed that serine-224 constitutes its C-terminus, and further analysis of the distribution of its monoisotopic masses by FAB-MS indicated that Ser224 possesses a carboxamide rather than a carboxylate group. Treatment of the 60 kDa cleavage fragment with cyanogen bromide and subsequent MALDI-TOF analysis of the partially RP-HPLC purified peptides yielded a fraction containing its N-terminal peptide. This peptide was digested with trypsin, and the digestion mixture was purified by HPLC. Analysis of the fractions by MALDI-TOF identified the N-terminal peptide and determined a mass of 2222. This mass suggested valine 226 was the N-terminal residue (modified by an adduct of 28 mass units). Larger amounts of the C-terminal tetrapeptide of the 60 kDa fragment (V226LIE229) were obtained by complete digestion of the crude reaction mixture with endoproteinase Glu-C. The peptide mixture was then purified on an immunoadsorbent column containing immobilized antibodies raised against a synthetic peptide with the sequence KVLIE. After elution of the affinity-bound peptide, it was analyzed by CID-MS verifying that an adduct of 28 mass units was attached to valine 226. These results indicated that the amino group of Val226 is formylated. The localization of the residues at the cleavage site of C225SR1 provides a biochemical identification of the active site region of the R1 subunit of RDPR from E.coli. The details of the mechanism of cleavage remain to be elucidated.

Assignment of free and disulfide-bonded cysteine residues in testis angiotensin-converting enzyme: functional implications.

Human testicular angiotensin-converting enzyme (tACE) is an extracellular protein that contains seven cysteine residues. The cysteines occur in a sequential distribution that is precisely mimicked in the tACE from rabbit and mouse, and in both domains of all known species of somatic ACE. One of the cysteines in human tACE, Cys496, is present in the reduced form as shown by labeling it with 5-[[2-(iodoacetyl)amino]ethylamino]naphthalene-1-sulfonic acid, isolating the fluorescent peptide from enzymatic digests by HPLC, and analyzing its sequence by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thiol reagents have no significant effect on the activity of tACE, indicating that this Cys is not involved in catalysis. The other six cysteines exist as three disulfides. Mass spectral analysis of cyanogen bromide peptides has established that the cystine connectivities follow a nearest-neighbor, aabbcc, pattern i.e., Cys152-Cys158, Cys352-Cys370, and Cys538-Cys550, in which the disulfides form three small loops of five, 17, and 11 residues, respectively. Although these disulfide loops constitute less than 5% of the total sequence of the protein, they contribute to the overall structural stabilization of tACE.

A human axillary odorant is carried by apolipoprotein D.

The characterization of the source of the odor in the human axillary region is not only of commercial interest but is also important biologically because axillary extracts can alter the length and timing of the female menstrual cycle. In males, the most abundant odor component is known to be E-3-methyl-2-hexenoic acid (E-3M2H), which is liberated from nonodorous apocrine secretions by axillary microorganisms. Recently, it was found that in the apocrine gland secretions, 3M2H is carried to the skin surface bound to two proteins, apocrine secretion odor-binding proteins 1 and 2 (ASOB1 and ASOB2) with apparent molecular masses of 45 kDa and 26 kDa, respectively. To better understand the formation of axillary odors and the structural relationship between 3M2H and its carrier protein, the amino acid sequence and glycosylation pattern of ASOB2 were determined by mass spectrometry. The ASOB2 protein was identified as apolipoprotein D (apoD), a known member of the alpha2mu-microglobulin superfamily of carrier proteins also known as lipocalins. The pattern of glycosylation for axillary apoD differs from that reported for plasma apoD, suggesting different sites of expression for the two glycoproteins. In situ hybridization of an oligonucleotide probe against apoD mRNA with axillary tissue demonstrates that the message for synthesis of this protein is specific to the apocrine glands. These results suggest a remarkable similarity between human axillary secretions and nonhuman mammalian odor sources, where lipocalins have been shown to carry the odoriferous signals used in pheromonal communication.

Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family.

The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.

Inactivation of inosine 5'-monophosphate dehydrogenase by the antiviral agent 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide 5'-monophosphate.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo guanine nucleotide biosynthesis. IMPDH converts inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with concomitant conversion of NAD+ to NADH. The antiviral agent 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) is believed to inhibit IMPDH by forming an active metabolite, the 5'-monophosphate EICARMP. The experiments reported here demonstrate that EICARMP irreversibly inactivates both human type II and Escherichia coli IMPDH. IMPDH is protected from EICARMP inactivation by IMP, but not by NAD+. Further, denaturation/renaturation of the EICARMP-inactivated enzyme did not restore enzyme activity, which indicates that EICARMP forms a covalent adduct with IMPDH. EICARMP was successfully used to titrate the active sites of IMPDH; these experiments demonstrate that four active sites are present in an IMPDH tetramer. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry of native E. coli IMPDH established that protein translation initiates at the third ATG of the DNA sequence. Thus, the E. coli IMPDH monomer is only 488 amino acids long and contains five instead of six cysteines. In addition, MALDI-TOF mass spectrometry showed that EICARMP is covalently bound to Cys-305 (Cys-331 in human type II IMPDH numbering), suggesting that Cys-305 functions as a nucleophile in the IMPDH reaction. The inactivation of the E. coli enzyme is a single-step reaction with kon = 1.94 x 10(4) M-1 s-1. In contrast, the inactivation of human type II IMPDH involves a two-step mechanism where Ki = 16 microM, k2 = 2.7 x 10(-2) s-1 and kon = 1.7 x 10(3) M-1 s-1. These results demonstrate that significant differences exist between bacterial and human IMPDH and suggest that this enzyme may be a target for antibiotic drugs.