Lars Hjelmqvist

ORMDL proteins are a conserved new family of endoplasmic reticulum membrane proteins

BackgroundAnnotations of completely sequenced genomes reveal that nearly half of the genes identified are of unknown function, and that some belong to uncharacterized gene families. To help resolve such issues, information can be obtained from the comparative analysis of homologous genes in model organisms.ResultsWhile characterizing genes from the retinitis pigmentosa locus RP26 at 2q31-q33, we have identified a new gene, ORMDL1, that belongs to a novel gene family comprising three genes in humans (ORMDL1, ORMDL2 and ORMDL3), and homologs in yeast, microsporidia, plants, Drosophila, urochordates and vertebrates. The human genes are expressed ubiquitously in adult and fetal tissues. The Drosophila ORMDL homolog is also expressed throughout embryonic and larval stages, particularly in ectodermally derived tissues. The ORMDL genes encode transmembrane proteins anchored in the endoplasmic reticulum (ER). Double knockout of the two Saccharomyces cerevisiae homologs leads to decreased growth rate and greater sensitivity to tunicamycin and dithiothreitol. Yeast mutants can be rescued by human ORMDL homologs.ConclusionsFrom protein sequence comparisons we have defined a novel gene family, not previously recognized because of the absence of a characterized functional signature. The sequence conservation of this family from yeast to vertebrates, the maintenance of duplicate copies in different lineages, the ubiquitous pattern of expression in human and Drosophila, the partial functional redundancy of the yeast homologs and phenotypic rescue by the human homologs, strongly support functional conservation. Subcellular localization and the response of yeast mutants to specific agents point to the involvement of ORMDL in protein folding in the ER.

The Alcohol Dehydrogenase System

Alcohol dehydrogenases of different types are common enzymes in nature. Two of these families, the medium-chain dehydrogenase/reductase family, MDR, and the shortchain dehydrogenase/reductase family, SDR, are well studied and known since long, but have experienced a recent “explosion” of new knowledge, extension and importance. The MDR family includes the classical zinc-containing liver alcohol dehydrogenases encompassing the classes of human liver alcohol dehydrogenase, while the SDR family includes the Drosophila alcohol dehydrogenase, which has shorter subunits, no similar metal requirements, other sub-domain arrangements with different structural relationships, and other subunit interactions.

Alcohol Dehydrogenase of Class IV (σσ-ADH) from Human Stomach

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, sigma sigma-ADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene, ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydrophobic, similar to those of the class I enzyme; this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, but this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in class IV by changes that may negatively affect coenzyme binding: Glu230, His271, Asn260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes.

Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain.

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.

Enzyme and Isozyme Developments within the Medium-Chain Alcohol Dehydrogenase Family

Alcohol dehydrogenases and related enzymes constitute a complex system of proteins derived from gene duplications at minimally four different levels. The system includes proteins of different type regarding family relationships and overall organiza tion. It also includes different enzymes within each family, as well as different classes of the enzymes, and different isozymes within the classes, apart from allelic variants. We have studied these relationships, starting with the horse liver alcohol dehydrogenase (Jornvall, 1970, Eklund et al., 1976), distinguishing the parallel evolution of separate enzyme types (Jornvall et al., 1981) and successively characterizing both the “medium-chain” (Jornvall et al., 1987) and “short-chain” (Persson et al., 1991) alcohol dehydrogenase families. Recently, we have characterized several novel forms, including both mammalian enzymes and those from other vertebrate lines, as well as from further, distantly related sources. Together, this has made it possible to deduce relationships of the functional and structural organization of the enzyme system, tracing gene duplications, original forms, and functional properties.

De novo sequencing of proteolytic peptides by a combination of C-terminal derivatization and nano-electrospray/ collision-induced dissociation mass spectrometry

A series of synthetic peptides (3–15 residues), C-terminally derivatized with 4-aminonaphthalenesulfonic acid (ansa), have been analyzed on a hybrid magnetic sector-orthogonal acceleration time-of-flight tandem mass spectrometer, fitted with a nano-electrospray (nano-ES) interface. Deprotonated molecules generated by negative-ion ES were subjected to collision-induced dissociation (CID) using either methane or xenon as the collision gas, at a collision energy of 400 eV (laboratory frame of reference). As a consequence of charge localization on the sulfonate group, only C-terminal fragment ions were formed, presumably by charge-remote fragmentation mechanisms. Interpretable CID spectra were obtained from fmol amounts of the small peptides (up to 6 residues), whereas low pmol amounts were required for the larger peptides. CID spectra were also recorded of derivatized, previously noncharacterised peptides obtained by proteolysis of cytosolic hamster liver aldehyde dehydrogenase. Interpretation of these CID spectra was based on rules established for the fragmentation of the synthetic peptides. This study shows that derivatization with ansa may be useful in the de novo sequencing of peptides.

Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis

N‐terminal acetylation of polypeptides is a common feature preventing direct Edman degradation. We describe a method for the removal of the acetyl group, with only a low extent of internal peptide bond cleavage, also in large proteins, by treatment at room temperature with trifluoroacetic acid and methanol. The alcohol is essential for selective deacetylation, and it is proposed that the deblocking mechanism consists of an acid‐catalyzed nucleophilic substitution involving methanol. The extent of deacetylation is limited, but the initial yield in the sequence analysis can be up to 10%. Deblocking of samples spotted or blotted onto sequencer filters is equally possible as the use of isolated samples from column separations. Deblocking on sequencer filters is also possible directly after negative results on initial sequencer attempts with samples proving to be blocked.

Multiplicity of N-terminal structures of medium-chain alcohol dehydrogenases Mass-spectrometric analysis of plant, lower vertebrate and higher vertebrate class I, II, and III forms of the enzyme

Ten different alcohol dehydrogenases, representing several classes of the enzyme and a wide spread of organisms, were analyzed for patterns of N‐terminal structures utilizing a combination of conventional and mass spectrometric peptide analysis. Results show all forms to be N‐terminally acetylated and allow comparisons of now 40 such alcohol dehydrogenases covering a large span of forms and origins. Patterns illustrate roles of acetylation in proteins in general, define special importance of the class I N‐terminal acetylation, and distinguish separate acetylated structures for all classes, as well as a common alcohol dehydrogenase motif.

Alcohol dehydrogenase of class III: consistent patterns of structural and functional conservation in relation to class I and other proteins☆

Class III alcohol dehydrogenase from the lizard Uromastix hardwickii has been characterized. This non‐mammalian, gnathostomatous vertebrate class III form allows correlations of structures and functions of this class, the traditional class I alcohol dehydrogenase, and other well‐studied proteins. Catalytically, results show similar recoveries and activities of all vertebrate class III forms independent of source, similar activities also in invertebrates but in lower amounts, and considerably higher specific activities in microorganisms. Structurally, variability patterns are consistent throughout the vertebrate system with a ratio in accepted point mutations versus class I of 0.4. This ratio between different classes of a zinc enzyme is comparable to that between different heme proteins (cytochrome c and myoglobin), suggesting defined but non‐identical functions also for the alcohol dehydrogenase classes.

Alcohol dehydrogenase of class I: kiwi liver enzyme, parallel evolution in separate vertebrate lines, and correlation with 12S rRNA patterns

Alcohol dehydrogenase class I from kiwi liver has been purified, analyzed, and compared with that of other alcohol dehydrogenases. The results show that several avian and mammalian forms of the enzyme exhibit parallel evolutionary patterns in two independent lineages of a single protein, establishing a pattern in common. Furthermore, the data correlate the enzyme evolutionary pattern with that of 12S rRNA. Biologically, the patterns complement those on ratite and other avian relationships. Functionally, the enzyme has a low K m with ethanol and a branched‐chain residue at position 141, like the mammalian enzymes but in contrast to the other characterized ratite enzyme (with Ala‐141 and a higher K m). This pattern of natural variability suggests a frequent but not fully complete correlation between a large residue size at position 141 and tight ethanol binding.