ALBERT

Description: The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). While formation and utilization of acyl-CoA takes place at the membrane, Acyl-CoA Binding Domain containing proteins (ACBD) in RBC cytosol bind acyl-CoA, limiting product feedback inhibition on ACSL and distribute Acyl-CoA to the various acyl-utilizing enzymes while protecting the cells against its potent detergent character. We have identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC, a protein with several isoforms that acts as a dimer. To relate its structure to activity, we report the expression of different modified forms in E. coli. Our data indicate that, despite the observed activity, enzyme studies of these mammalian membrane proteins in the host E. coli are strongly hampered by their aggregation into inclusion bodies. While activity can be measured, data on enzyme kinetics and specificity are questionable. Oleoyl-CoA formation from oleic acid, CoA and ATP reveled that the two transmembrane spanning segments predicted at the amino-terminus of the protein are not essential for its activity. Moreover, they are not essential for dimer formation and strong association with membranes. ACSL6 appears to be an integral membrane protein. One of the five spliced isoforms of ACSL6 reported, lacks the so-called fatty acid Gate-domain, and appears to be unable to activate long chain fatty acids. We hypothesize that this form modulates activity of the other active isoforms of ACSL6 through hetero-dimer formation. An EST clone of erythroid precursor cells identified ACBD6 as a potential AcylCoA binding protein in RBC. This modular protein contains an Acyl-CoA binding domain at the amino-terminus and two Ankyrin-repeat motifs (ANK) at the C-terminus. Both the full-length protein and the N-terminus domain were soluble when expressed and purified in E. coli. Expression of the C-terminus domain by itself rendered an insoluble protein. We report that ACBD6 binds long-chain acyl-CoA with a preference for C18:1-CoA over C20:4 and C16:0-CoA and does not bind fatty acid. Truncation of the ANK domain had no effect on the binding activity of the N-terminus domain. Together our findings implicate ACBD6 as part of the Acyl-CoA turnover mechanism in RBC, its actual role on the kinetics of ACSL and/or LPLAT activity needs to be established. The description of these proteins involved in Acyl-CoA turnover in RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.

Description: The formation, distribution and utilization of acyl-CoA plays a crucial role in plasma membrane phospholipid turnover in red blood cells (RBC). Upon de-acylation of glycero-phospholipids (PL) via the action of phospholipase, re-acylation of the lysophospholipids (LPL) requires activity of two enzymes of the Lands pathway. Long-chain acyl-CoA synthetases (ACSL) activate fatty acids to acyl-CoA which are subsequently ligated to LPL by LysoPhosphoLipid Acyl Transferase (LPLAT) a family of enzymes with exclusive specificity for the polar group of LPL (phosphatidic acid, choline, serine and ethanolamine). We recently identified ACSL6 as the enzyme responsible for the activation of fatty acid in RBC. None of the family members of LPLAT have been identified in RBC to date. LPC, either generated in the RBC or taken up from plasma, is rapidly acylated by RBC suggesting an important role for Lysohosphatidylcholine-acyl transferase (LPCAT) in RBC. We report the identification and characterization of LPCAT, the enzyme that generates PC from LPC and acylCoA. We identified the RNA expression of LPCAT, an approximately 60kD protein, in reticulocytes, confirming proteomic studies suggesting the presence of this protein in adult RBC membranes.). It is a modular protein containing an acyltransferase domain at the amino-terminus, three predicted membrane spanning domains, and a putative calcium binding site at the C-terminus, distinguishing it from the lysophosphatidic acid acyltransferease (LPAAT). The putative LPCAT was expressed in E. coli. It was found in the E. coli membrane fraction, and was able to use oleoyl-CoA and LPC as substrates to generate PC. Lysophosphatidic acid (LPA) was not acylated by this protein. In contrast the previously identified LPAAT (1) expressed in E. coli, utilized LPA but not LPC, indicating LPL specificity of these enzymes. Radioactive fatty acid added to RBC is also incorporated in phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). Sequence analysis suggests that two other proteins present in the genome of mammals are homologues of LPCAT. We hypothesize that these putative acyltransferases are responsible for the acylation of lysophosphatidyl ethanolamine (LPE) and lysophosphatidyl serine (LPS). These proteins are essential to maintain a proper glycerophospholipid composition of the RBC membrane and thereby viability of the cells. A dysfunction of this system may underlie the observed differences in phospholipid molecular species composition in subpopulations of sickle cells contributing to sickle cell pathology. A complete description of these proteins involved in the maintenance of glycerophospholipid composition of RBC will aid to better understand the maintenance of plasma membrane lipid composition of all mammalian cells.

Description: In mammals, long-chain acyl-CoA synthetases (ACSL) are necessary for fatty acid degradation, phospholipid remodeling, and production of long acyl-CoA esters that regulate various physiological processes. These enzymes play a crucial role in plasma membrane phospholipid turnover in erythrocytes, maintaining the complex phospholipid molecular species composition essential for proper membrane function. The mechanism by which this highly dynamic turnover together with an ever-changing plasma fatty acid pool maintains phospholipid composition is poorly understood. We have previously cloned Acyl-CoA Synthetase Long-chain member 6 (ACSL6), the isoform responsible for activation of long-chain fatty acids in erythrocytes. Two additional transcript variants of this protein were subsequently isolated from brain and testis. We report the expression of four different variants of ACLS6 in reticulocytes, one as we originally reported, two of which are novel, one as was identified in brain cells. PCR amplifications using primers for the predicted variable regions were performed from cDNAs of CD34 positive erythroid progenitors, K562 cells, fetal blood cells, reticulocytes and placenta. ACSL variants were expressed in E. coli host BL21DE3 cells using the pET28a vector, and detected by His tag immuno detection. Sequence alignments were generated using sequences retrieved from RefSeq and GenBank databases on the NCBI site. Exon and intron definition for ACSL members were obtained using evidence viewer and model maker available at the map viewer page of each gene. We identified four different spliced variants of ACSL6 in erythroid cells based on a mutually exclusive exon pair. Each exon of this pair encodes a slightly different short motif that contains the fatty acid Gate domain, a conserved structural domain found in all vertebrate and invertebrate ACSL homologs. The motif differs in the presence of either the aromatic residue phenylalanine (Phe) or tyrosine (Tyr), and seems to play a role in substrate specificity. One of the new forms contained an exon not found in any other ACSL isoforms. Erythroid precursors also express the closely related ACSL1, and we characterized two additional isoforms of this protein, similar to ACSL6. When analyzed on denaturing SDS polyacrylamide gel both ACSL1 and 6 appeared to exist as a dimer. Based on our results, we propose the generation of two different Gate-domains by alternative splicing of the two exons in these proteins. One represents a switch of the Phe to the Tyr Gate-domain motif, the other resulted of the exclusion of both. Swapping of this motif appears to be common to all mammalian homologs of ACSL1 and 6. We conclude that the Phe to a Tyr substitution in the Gate-domain, or its removal, together with the formation of homo or heterodimers will allow ACSL6 the structural diversity to define substrate specificity that maintains the complex plasma membrane phospholipid molecular species composition in erythrocytes.