G6b, a novel immunoglobulin superfamily member encoded in the human major histocompatibility complex, interacts with SHP-1 and SHP-2.

The G6b gene, located in the class III region of the human major histocompatibility complex, has been suggested to encode a putative receptor of the immunoglobulin superfamily. Genomic sequence information was used as a starting point to clone the corresponding cDNA. Reverse transcriptase polymerase chain reaction showed that expression of the gene is restricted to certain hematopoietic cell lines including K562, Molt 4, and Jurkat. Several splice variants were detected, varying only in their C-terminal parts. One of the potential membrane-bound isoforms contained two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Four of the isoforms were expressed as epitope-tagged proteins in the cell lines K562 and COS-7. The two splice isoforms lacking the hydrophobic transmembrane segment were secreted from the cell. Glycosidase treatment of the four recombinant proteins provided evidence for N- and O-glycosylation. Immunofluorescence studies indicated that the spliced isoforms having a transmembrane segment were directed to the cell membrane. The G6b isoform containing two immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail was found to be phosphorylated on tyrosine residues after pervanadate treatment of cells and, subsequently, interacts with the SH2-containing protein-tyrosine phosphatases SHP-1 and SHP-2. Mutagenesis studies showed that phosphorylation of tyrosine 211 is critical for the interaction of G6b with SHP-1 and SHP-2.

Alkyl-dihydroxyacetonephosphate synthase. Presence and role of flavin adenine dinucleotide.

Alkyl-dihydroxyacetonephosphate synthase is a peroxisomal enzyme involved in ether lipid synthesis. It catalyzes the exchange of the acyl chain in acyl-dihydroxyacetonephosphate for a long chain fatty alcohol, yielding the first ether linked intermediate, i.e. alkyl-dihydroxyacetonephosphate, in the pathway of ether lipid biosynthesis. Although this reaction is not a net redox reaction, the amino acid sequence of the enzyme suggested the presence of a flavin adenine dinucleotide (FAD)-binding domain. In this study we show that alkyl-dihydroxyacetonephosphate synthase contains an essential FAD molecule as cofactor, which is evidenced by fluorescence properties, UV-visible absorption spectra and the observation that the enzyme activity is dependent on the presence of this cofactor in a coupled in vitro transcription/translation assay. Furthermore, we could demonstrate that the FAD cofactor directly participates in catalysis. Upon incubation of the enzyme with the substrate palmitoyl-dihydroxyacetonephosphate, the flavin moiety is reduced, indicating that in this initial step the substrate is oxidized. Stopped flow experiments show that the reduction of the flavin moiety is a monophasic process yielding a oxygen stable, reduced enzyme species. Upon addition of hexadecanol to the reduced enzyme species, the flavin moiety is efficiently reoxidized. A hypothetical reaction mechanism is proposed that is consistent with the data in this paper and with previous studies.

Alkyl-dihydroxyacetonephosphate synthase.

The initial steps of ether phospholipid biosynthesis take place in peroxisomes. Alkyl-dihydroxyacetonephosphate synthase, the peroxisomal enzyme that actually introduces the ether linkage, has been purified from guinea pig liver in this laboratory. With the amino acid sequences obtained from this protein, the authors were able to clone the cDNAs encoding this enzyme from both guinea pig and human liver. In both cases, the enzyme appears to be synthesized as a precursor protein with a N-terminal cleavable presequence containing a peroxisomal targeting signal (PTS) type 2. Levels of the enzyme protein were found to be strongly reduced in human fibroblasts derived from Zellweger syndrome and rhizomelic chondrodysplasia punctata patients. The molecular basis of an isolated alkyl-dihydroxyacetonephosphate synthase deficiency was resolved. A clone encoding a Caenorhabditis elegans homolog of the mammalian enzymes was characterized. In contrast to the mammalian enzymes, this C. elegans enzyme lacks a N-terminal PTS type 2 motif, but carries a C-terminal PTS type 1.

Ether lipid biosynthesis: alkyl-dihydroxyacetonephosphate synthase protein deficiency leads to reduced dihydroxyacetonephosphate acyltransferase activities.

Recent studies have indicated that two peroxisomal enzymes involved in ether lipid synthesis, i.e., dihydroxyacetonephosphate acyltransferase and alkyl-dihydroxyacetonephosphate synthase, are directed to peroxisomes by different targeting signals, i.e., peroxisomal targeting signal type 1 and type 2, respectively. In this study, we describe a new human fibroblast cell line in which alkyl-dihydroxyacetonephosphate synthase was found to be deficient both at the level of enzyme activity and enzyme protein. At the cDNA level, a 128 base pair deletion was found leading to a premature stop. Remarkably, dihydroxyacetonephosphate acyltransferase activity was strongly reduced to a level comparable to the activities measured in fibroblasts from patients affected by the classical form of rhizomelic chondrodysplasia punctata (caused by a defect in peroxisomal targeting signal type 2 import). Dihydroxyacetonephosphate acyltransferase activity was completely normal in another alkyl-dihydroxyacetonephosphate synthase activity-deficient patient. Fibroblasts from this patient showed normal levels of the synthase protein and inactivity results from a point mutation leading to an amino acid substitution. These results strongly suggest that the activity of dihydroxyacetonephosphate acyltransferase is dependent on the presence of alkyl-dihydroxyacetonephosphate synthase protein. This interpretation implies that the deficiency of dihydroxyacetonephosphate acyltransferase (targeted by a peroxisomal targeting signal type 1) in the classic form of rhizomelic chondrodysplasia punctata is a consequence of the absence of the alkyl-dihydroxyacetonephosphate synthase protein (targeted by a peroxisomal targeting signal type 2).

Characterization of recombinant guinea pig alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli. Kinetics, chemical modification and mutagenesis.

A recombinant form of guinea pig alkyl-dihydroxyacetonephosphate synthase, a key enzyme in the biosynthesis of ether phospholipids, was characterized. Kinetic analysis yielded evidence that the enzyme operates by a ping-pong rather than a sequential mechanism. Enzyme activity was irreversibly inhibited by N-ethylmaleimide, p-bromophenacylbromide and 2,4-dinitrofluorobenzene. The enzyme could be protected against the inactivation by either of these three compounds by the presence of saturating amounts of the substrate palmitoyl-dihydroxyacetonephosphate. The rate of inactivation of the enzyme by p-bromophenacylbromide was strongly pH dependent and the highest at alkaline conditions. Collectively, these results are indicative of cysteine, histidine and lysine residues, respectively, at or close to the active site. The divalent cations Mg2+, Zn2+ and Mn2+ were found to be inhibitors of enzymatic activity, whereas Ca2+ had no effect. Mutational analysis showed that histidine 617 is an essential amino acid for enzymatic activity: replacement of this residue by alanine resulted in complete loss of enzymatic activity. A recombinant enzyme with the C-terminal five amino acids deleted was shown to be inactive, indicating an important role of the C-terminus for catalytic activity.

Nucleotide sequence of alkyl-dihydroxyacetonephosphate synthase cDNA from Dictyostelium discoideum.

The nucleotide sequence is reported of alkyl-dihydroxyacetonephosphate synthase cDNA from the cellular slime mold Dictyostelium discoideum. The open reading frame encodes a protein of 611 amino acids which shows a 33% amino acid identity to the human enzyme. This D. discoideum homolog carries a variant of the peroxisomal targeting signal type 1 at its C-terminus (PKL). Expression of the cDNA in Escherichia coli yielded an enzymatically active protein.

Alkyl-dihydroxyacetonephosphate synthase. Fate in peroxisome biogenesis disorders and identification of the point mutation underlying a single enzyme deficiency.

Peroxisomes play an indispensible role in ether lipid biosynthesis as evidenced by the deficiency of ether phospholipids in fibroblasts and tissues from patients suffering from a number of peroxisomal disorders. Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme playing a key role in the biosynthesis of ether phospholipids, contains the peroxisomal targeting signal type 2 in a N-terminal cleavable presequence. Using a polyclonal antiserum raised against alkyl-dihydroxyacetonephosphate synthase, levels of this enzyme were examined in fibroblast cell lines from patients affected by peroxisomal disorders. Strongly reduced levels were found in fibroblasts of Zellweger syndrome and rhizomelic chondrodysplasia punctata patients, indicating that the enzyme is not stable in the cytoplasm as a result of defective import into peroxisomes. In a neonatal adrenoleukodystrophy patient with an isolated import deficiency of proteins carrying the peroxisomal targeting signal type 1, the precursor form of alkyl-dihydroxyacetonephosphate synthase was detected at a level comparable to that of the mature form in control fibroblasts, in line with an intraperoxisomal localization. A patient with an isolated deficiency in alkyl-dihydroxyacetonephosphate (DHAP) synthase activity had normal levels of this protein. Analysis at the cDNA level revealed a missense mutation leading to a R419H substitution in the enzyme of this patient. Expression of a recombinant protein carrying this mutation in Escherichia coli yielded an inactive enzyme, whereas a comparable control recombinant enzyme was active, providing further proof that this substitution is responsible for the inactivity of the enzyme and the phenotype. In line with this result is the observation that wild-type alkyl-DHAP synthase activity can be inactivated by the arginine-modifying agent phenylglyoxal. The enzyme is efficiently protected against this inactivation when the substrate palmitoyl-DHAP is present at a saturating concentration. The gene encoding human alkyl-dihydroxyacetonephosphate synthase was mapped on chromosome 2q31.

Nucleotide sequence of a cDNA clone encoding a Caenorhabditis elegans homolog of mammalian alkyl-dihydroxyacetonephosphate synthase: evolutionary switching of peroxisomal targeting signals.

The nucleotide sequence is reported of a cDNA clone encoding a Caenorhabditis elegans homolog of guinea pig and human alkyl-dihydroxyacetonephosphate synthase. The open reading frame encodes a protein of 597 amino acids which shows extensive homology with the mammalian enzymes (52% identical and about 76% similar in the overlapping region). In contrast to the mammalian enzymes, which carry a consensus peroxisomal targeting signal type 2 in a cleavable N-terminal presequence, this Caenorhabditis elegans homolog carries a consensus peroxisomal targeting signal type 1 (CKL) at its C-terminus. Expression of this protein in an in vitro transcription/translation system yielded a 65 kDa protein. Recombinant aenorhabditis elegans alkyl-DHAP synthase expressed in the yeast Pichia pastoris was enzymatically active.

Alkyl-dihydroxyacetonephosphate synthase.

Mammalian ether phospholipids are characterized by a glycero-ether linkage at the sn-1-position of the glycerol backbone. In humans this type of phospholipid species occurs mainly in the ethanolamine and choline phosphoglycerides comprising an estimated 15% of total phospholipids. The glycero-ether linkage is synthesized by replacement of the acyl chain in acyl-dihydroxyacetonephosphate by a long-chain alcohol that donates the oxygen for the ether linkage. Both the enzyme that forms acyl-dihydroxyacetone phosphate (see Chapter II of this volume) and the one that introduces the glycero-ether linkage. i.e. alkyl-dihydroxyacetonephosphate synthase, are located in peroxisomes. The deficiency of ether phospholipids in human inborn errors of metabolism, caused by defects in peroxisome biogenesis, has clearly delineated the indispensable role of peroxisomes in ether phospholipid synthesis. The most characteristic enzyme of ether lipid synthesis is alkyl-dihydroxyacetonephosphate synthase. Its discovery and some of its properties, including mechanistic studies, have been discussed in recent reviews. This review recapitulates these findings and focuses on the new insights into the structure and properties of the enzyme that have recently been obtained resulting from the purification and subsequent cloning and expression of the cDNA encoding this peroxisomal enzyme.

Immunological localization and tissue distribution of alkyldihydroxyacetonephosphate synthase and deficiency of the enzyme in peroxisomal disorders.

Alkyldihydroxyacetonephosphate synthase (alkylglycerone-phosphate synthase) is a peroxisomal enzyme involved in ether phospholipid biosynthesis. The recent cloning of the cDNA encoding this enzyme from guinea pig liver enabled the raising of specific antisera against this enzyme. Both a synthetic peptide corresponding to a predicted epitope and a recombinant protein expressed in Escherichia coli were used for that purpose. Using western blot techniques, the solubilization of the enzyme from the peroxisomal membrane by Triton X-100 in the presence of salt was confirmed. Neutral hydroxylamine treatment of peroxisomes resulted in almost no release of the protein from the membrane. The complete polypeptide chain of the enzyme was resistant to proteolysis by trypsin when intact peroxisomes were studied. Carbonate treatment released alkyldihydroxyacetonephosphate synthase from the membrane indicating that the enzyme is not an integral membrane protein. This idea is in accord with the absence of a clear hydrophobic transmembrane domain in the deduced amino acid sequence of the enzyme. Alkyldihydroxyacetonephosphate synthase, as well as its mRNA, could be detected in all five guinea pig tissues examined. When using the antiserum against guinea pig recombinant alkyldihydroxyacetonephosphate synthase, a cross-reactive protein was detected in a human liver homogenate that runs at a slightly higher molecular mass. The absence of this band in liver of Zellweger syndrome and Rhizomelic chondrodysplasia punctata patients provides strong evidence that it represents the human homolog of this enzyme.

Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a peroxisomal targeting signal 2.

Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes. The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98%. The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme. Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information. This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase. One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence. An unexpected homology with D-lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed.

Polymerase chain reaction-based cloning of alkyl-dihydroxyacetonephosphate synthase complementary DNA from guinea pig liver.

Peroxisomes are indispensable organelles for ether lipid biosynthesis in mammalian tissues, and the deficiency of these organelles in a number of peroxisomal disorders leads to deficiencies in ether phospholipids. We have previously purified the committed enzyme for ether lipid biosynthesis, i.e. alkyl-dihydroxyacetone-phosphate synthase, to homogeneity. We have now determined the N-terminal amino acid sequence, as well as additional internal sequences obtained after cyanogen bromide cleavage of the enzyme. With primers directed against the N-terminal sequence and against a cyanogen bromide fragment sequence, a 1100-bp cDNA fragment was obtained by conventional polymerase chain reaction using first-strand cDNA from guinea pig liver as a template. The 5' and 3' ends of the cDNA were obtained by rapid amplification of cDNA ends. The open reading frame encodes a protein of 658 amino acids, containing the N-terminal amino acid sequence as well as the cyanogen bromide cleavage fragment sequences. The derived amino acid sequence includes a mature protein 600 amino acids long and a presequence 58 amino acids long. The latter contains a stretch of amino acids known as peroxisomal targeting signal 2. The size of the mRNA was estimated to be around 4200 nucleotides. Recombinant His-tagged alkyl-dihydroxyacetonephosphate synthase expressed in Escherichia coli was enzymatically active.