Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene

Previously we identified and cloned the cDNA for a new protein, apolipoprotein L (apoL), present in plasma and mainly associated with large high density lipoprotein particles. Using 5′ rapid amplification of cDNA ends, RT-PCR and comparison with three Human Genome Project and three expressed sequence tag sequences, we have characterized the gene for apoL and for three additional, highly homologous proteins that constitute a new family of proteins that display no homology with previously described apolipoproteins. The genes for all four proteins, apoL-I, apoL-II, apoL-III, and apoL-IV, are located at chromosome 22q12.1-13.1 within a 127,000-bp region. The apoL-I gene is in the opposite orientation to the other three. All four genes have TATA-less promoters, which contain putative sterol regulatory elements, suggesting that transcription of these genes may be coordinated with that of the low density lipoprotein receptor and genes in pathways involving the synthesis of triglycerides and cholesterol. The gene family has a consensus eight-exon structure with alternative splice sites that could produce as many as eight distinct gene products. The apoL-II and apoL-III genes have alternative transcriptional start sites as a result of additional 5′ exons. apoL-I, apoL-II, and apoL-III are expressed to the highest degree in the lung. Other tissues with high expression are the pancreas, prostate, spleen, liver, and placenta. Four clustered common polymorphisms, three of which altered the protein sequence, were found in apoL-I, all in linkage disequilibrium, and describing two haplotypes: the more common Lys166/Ile244/Lys271 and the rarer Glu166/Met244/Arg271. —Duchateau, P. N., C. R. Pullinger, M. H. Cho, C. Eng, and J. P. Kane. Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene. J. Lipid Res. 2001. 42: 620–630. Previously we identified and cloned the cDNA for a new protein, apolipoprotein L (apoL), present in plasma and mainly associated with large high density lipoprotein particles. Using 5′ rapid amplification of cDNA ends, RT-PCR and comparison with three Human Genome Project and three expressed sequence tag sequences, we have characterized the gene for apoL and for three additional, highly homologous proteins that constitute a new family of proteins that display no homology with previously described apolipoproteins. The genes for all four proteins, apoL-I, apoL-II, apoL-III, and apoL-IV, are located at chromosome 22q12.1-13.1 within a 127,000-bp region. The apoL-I gene is in the opposite orientation to the other three. All four genes have TATA-less promoters, which contain putative sterol regulatory elements, suggesting that transcription of these genes may be coordinated with that of the low density lipoprotein receptor and genes in pathways involving the synthesis of triglycerides and cholesterol. The gene family has a consensus eight-exon structure with alternative splice sites that could produce as many as eight distinct gene products. The apoL-II and apoL-III genes have alternative transcriptional start sites as a result of additional 5′ exons. apoL-I, apoL-II, and apoL-III are expressed to the highest degree in the lung. Other tissues with high expression are the pancreas, prostate, spleen, liver, and placenta. Four clustered common polymorphisms, three of which altered the protein sequence, were found in apoL-I, all in linkage disequilibrium, and describing two haplotypes: the more common Lys166/Ile244/Lys271 and the rarer Glu166/Met244/Arg271. —Duchateau, P. N., C. R. Pullinger, M. H. Cho, C. Eng, and J. P. Kane. Apolipoprotein L gene family: tissue-specific expression, splicing, promoter regions; discovery of a new gene. J. Lipid Res. 2001. 42: 620–630. Epidemiological studies have shown that individuals with low levels of high density lipoproteins (HDL) in blood have a substantially increased risk of coronary artery disease (1Gordon T. Cotelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. High density lipoprotein as a protective factor against coronary heart disease: the Framingham Study.Am. J. Med. 1977; 66: 707-714Google Scholar, 2Miller G.S. Miller N.F. Plasma high density lipoprotein and development of ischemic heart disease.Lancet. 1975; 1: 16-19Google Scholar). Mechanisms by which HDL protect against atherosclerosis involve reverse cholesterol transport (3Bruce C. Chouinard Jr., R.A. Tall A.R. Plasma lipid transfer proteins, high-density lipoproteins, and reverse cholesterol transport.Ann. Rev. Nutr. 1998; 18: 297-330Google Scholar), whereby HDL acquire cholesterol from peripheral cells and facilitate its esterification and delivery to the liver, and the ability of HDL to impede the oxidation of other plasma lipoproteins (4Kunitake S.T. Jarvis M. Hamilton R.L. Kane J.P. Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins.Proc. Natl. Acad. Sci. USA. 1992; 89: 6993-6997Google Scholar, 5Parthasarathy S. Barnett J. Fong L.G. High-density lipoprotein inhibits the oxidative modification of low-density lipoprotein.Biochim. Biophys. Acta. 1990; 1044: 275-283Google Scholar). Other protective roles may be revealed by identifying novel protein constituents of HDL (6James R.W. Hochstrasser D. Tissot J-D. Funk M. Appel R. Barja F. Pellegrini C. Muller A.F. Pometta D. Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotien A-II isolated from human plasma.J. Lipid Res. 1988; 29: 1557-1571Google Scholar, 7Kunitake S.T. Carilli C.T. Lau K. Protter A.A. Naya-Vigne J. Kane J.P. Identification of proteins associated with apolipoprotein A-I-containing lipoproteins purified by selected-affinity immunosorption.Biochemistry. 1994; 33: 1988-1993Google Scholar). Because ultracentrifugation causes protein dissociation and can modify HDL structures, we used a purification strategy that conserves lipoprotein integrity: selected affinity immunosorption, which isolates lipoproteins under minimally perturbing conditions (8McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I (high density lipoproteins).Proc. Natl. Acad. Sci. USA. 1984; 81: 1356-1360Google Scholar). Using this technique, combined with two-dimensional electrophoresis, we isolated apolipoprotein L (apoL), a novel 42-kDa protein (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar), together with a 38-kDa truncated form of apoL. Selected affinity isolation, using an antibody to apoL, showed the protein to be associated chiefly with large HDL particles (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar). Secondary structure analysis of the protein sequence, deduced from the cDNA sequence, revealed the presence of four lipid-binding amphipathic α-helices. Computer-based homology searches revealed no significant similarity with other proteins. Furthermore, plasma levels of apoL were shown to be correlated with total plasma triglycerides and cholesterol in normolipidemic subjects, whereas triglycerides were the major independent covariate influencing apoL concentration in plasma of dislipidemic patients and those with type 2 diabetes (10Duchateau P.N. Movsesyan I. Schoenhaus S.A. Spencer S.J. Mark S. Jaffe R.B. Redberg R.F. Kane J.P. Malloy M.J. Plasma apolipoprotein L levels are influenced by plasma triglyceride levels in normolipidemic, hyperlipidemic and NIDDM subjects.J. Lipid Res. 2000; 41: 1231-1236Google Scholar). In the present study we have determined the 5′ end of apoL (now termed apoL-I) mRNA using 5′-rapid amplification of cDNA ends (RACE). By reverse transcriptase PCR we have found an alternatively spliced variant containing an additional exon. These studies revealed the presence of apoL-I gene polymorphisms, the frequencies of which were studied among a group of control individuals. In the course of our studies, clones for three closely related cDNAs were obtained and analyzed. Using Internet-based Basic Local Alignment Search Tool (BLAST) searches and comparison with the sequences of these cDNAs, we have studied the structure of a family of four apoL-related genes. The locations of the promoter regions were determined. These were examined for regulatory elements that potentially control expression of the apoL genes. Sites of expression of the apoL genes were determined by establishing a competitive quantitative RT-PCR assay and by dot blotting. We compared our previously reported apoL-I cDNA sequence (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar) and the additional 5′ sequence of apoL-I that we determined by 5′ RACE with GenBank entries using BLAST at http://www.ncbi.nlm.nih.gov. Primers were designed using the sequences with accession numbers Z95144 and Z82215, and PCR was carried out using human genomic DNA. These PCR products were analyzed to confirm the sequences of the intron-exon junctions predicted from a comparison of these genomic sequences and our cDNA clones. Full-length cDNA clones for apoL-I, apoL-II, apoL-III, and apoL-IV were generated by RT-PCR (see below). DNA sequencing was performed directly on PCR products or cDNA clones using the Thermo Sequenase Cycle Sequencing kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ) with primers end-labeled with γ-33PATP (Amersham). The extent of the homology between the corresponding exons of the four genes was compared using the GeneWorks program (Oxford Molecular Group, Inc., Campbell, CA). Clustal alignment analysis of the peptide sequences was performed using the MacVector program (Oxford Molecular Group). To determine the 5′ untranslated portion of the apoL-I mRNA, we performed conventional 5′ RACE using two primers designed from our previously reported cDNA sequence (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar). Primer #453 (Table 1) was used to make the first strand cDNA from human pancreatic poly A+ RNA using a 5′/3′ RACE kit (Boehringer Mannheim Corp., Indianapolis, IN). Primer #449 (Table 1), together with the oligo d(T)-anchor primer, was used for the RACE reaction. Nested PCR was performed using primer (Table 1) and the PCR products were cloned and the clones as described primers were designed from and sequences, revealed by these studies, and from homologous regions in the three other genes. These were used for 5′ RACE studies, using the The sequence of apoL-III, found using primers and (Table 1), was used to primers and (Table These were used in 5′ RACE to determine the 5′ end of primers used in this of of and of of and of of and of exons and of of of of of the primer that the primer is and the is in in the in a new the primer that the primer is and the is in in the primer, (Table 1), in that to apoL-I, and was used to cDNA from human poly A+ RNA using the cDNA amplification kit Inc., CA). The cDNA was to the cDNA and to PCR with the primer and with primer and or using the cDNA PCR kit protein sequences were analyzed for the presence of and the of H. J. S. Identification of and and of 1997; Scholar) human RNA was with an apoL-I to the dot of purified poly A+ RNA from of human The for the expression of eight genes. from the 5′ end of of apoL-I was generated by using primers and and with by using the DNA (Amersham). The was to and the was and analyzed on a using the program at of apoL-I and apoL-II identified by 5′ primer was with in of apoL-I to These two primers were designed that to the homologous sequences in apoL-I and and reverse transcriptase (Boehringer were used to PCR products were cloned and to the apoL-I and apoL-II In studies, many of these clones were from a homologous which we To determine the of the apoL-III splice in RT-PCR products that were with for apoL-I, apoL-II, or These were and The reverse transcriptase were carried out at for using of total RNA from eight tissues in a total of containing (Boehringer reverse transcriptase (Boehringer and at The PCR were carried out in the in a total of in of and of primer and The were for at and to for for and for Full-length apoL-IV clones were obtained by RT-PCR using the this two primers were designed and 1) for apoL-IV from a comparison of the and the untranslated sequences of the four genes. To the of apoL-I, apoL-II, and apoL-III mRNA in two primers and 1) that to homologous sequences in the of gene were used for quantitative The RNA used in these was generated from an apoL-I PCR using RNA (Boehringer PCR was from an apoL-I using a primer and primer which was designed to a containing a In the RT-PCR the RNA a To the of the two of control were carried out as described by and J. M. C. of quantitative reverse transcriptase PCR used to expression of and genes in J. 1997; Scholar). In a of was used with of total In the other the and total of RNA were and the of was The RT-PCR conditions were as described with of total RNA used for comparison of generated on the from the using Search C. of the and of of Scholar) and by K. K. H. T. and new and for of consensus in sequence Res. Scholar) was to in the 5′ regulatory elements that potentially control the expression of the apoL genes. We a of sequence between our apoL-I cDNA and Z82215, in the corresponding to These were by this using primers and by using genomic DNA from control PCR conditions were as described that were for for and for These PCR products were directly using the Thermo Sequenase Cycle Sequencing kit (Amersham). additional genomic DNA were analyzed for the and by previously reported apoL-I cDNA sequence, together with the additional 5′ sequence determined by 5′ was compared with GenBank entries using These searches found three expressed sequence of exons and and two of and We with the of a a in the genomic sequence All of the was in and these were examined by PCR and sequencing from individuals. a a of four was within a (Table the predicted protein is a and an These were studied in additional control individuals to determine more the frequencies (Table These two are in linkage The more sequencing the to be in linkage with the and we two haplotypes: the more common Lys166/Ile244/Lys271 and the common Glu166/Met244/Arg271. In the sequence of the apoL-I protein is from the Lys166/Ile244/Lys271 and and in a new of the four apoL gene family proteins. or The of apoL-I, apoL-III, and apoL-IV to the apoL-III and apoL-IV, the three and two are shown In the of the is by amphipathic are and are The apoL-I sequence shown the common of two is the common Glu166/Met244/Arg271. These are by other found the sequences in the individuals showed with the sequence and from our was and was These are in with in the we found in to be as we and as in and genomic sequences for two additional genes and with homology to apoL-I were in and and the present studies were the sequence of the of human chromosome was reported I. S. A.R. R. M. L.J. R. J.P. C. J. K. S. D. J. DNA sequence of human chromosome Scholar). The the presence of apoL-I sequences, for that of the peptide by exons 2 and and the identified a homologous gene and apoL-II, as we The regions corresponding to exons and the of of apoL-II were identified by the I. S. A.R. R. M. L.J. R. J.P. C. J. K. S. D. J. DNA sequence of human chromosome Scholar). of the gene for a protein H. are to in are expressed in of and two novel Scholar) was identified in the chromosome I. S. A.R. R. M. L.J. R. J.P. C. J. K. S. D. J. DNA sequence of human chromosome Scholar). The mRNA sequence for this protein to of and exons of the apoL-III gene. is of the we and the peptide of We to the apoL-III to be with the of apoL-I and The start sequences in and are in a and that in in a to for of M. cDNA from studies on Scholar). searches revealed an containing and of exons and of The apoL-III gene for and orientation of the apoL gene family on chromosome The of the is as is that of the type the analysis of these genes and apoL-I by we clones from a which we have on and was we were to the gene for apoL-IV on this of the apoL-IV sequence predicted two to the as a result of present or These proteins, are and The corresponding mRNA sequences have to The accession numbers are and The apoL-IV 2 is homologous to of apoL-I, whereas of apoL-IV is The start in is in and that in 2 is in an M. cDNA from studies on Scholar). The sequences for all of the exons described in have to The accession numbers are apoL-I, apoL-II, apoL-III, and apoL-IV, additional sequence was found by 5′ RACE of the of our cDNA for apoL-I (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar). of the sequence with the human chromosome sequence from revealed that apoL-I gene and exons The sequences the predicted intron-exon for apoL-I were by and sequencing using primers designed from the of clones from the 5′ RACE that 2 was present in an in an with M. cDNA from studies on Scholar). The presence of 2 a protein with a peptide The common a protein with a peptide of more we reported (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar). The accession numbers of the two apoL-I mRNA sequences and are and the combined 5′ RACE for the four apoL genes. the clones are and is shown in total of clones was for The transcription start sites for all four genes. with the low to of the this is with the to by primer analysis clones with were found for apoL-III and for The apoL-III clones and of 5′ RACE studies were performed to the 5′ end of apoL-III is and from to is and from to of apoL-III containing exons and have a of from to The accession numbers of these two apoL-III mRNA sequences and are and of the apoL-II revealed which for from to The accession corresponding to this is and that of the common is the apoL-II is from to and the splice at is homologous to that of apoL-III gene at this splice consensus sequence is (see in the apoL-I or apoL-IV and no was found for alternative in these other two genes. The of the apoL genes were by a of and We that the for apoL-I to the at The of apoL-II and apoL-III are with two apoL-II these are and of the at The corresponding for apoL-III are and and the is at are four in the corresponding of the apoL-IV gene. We by that the of at was a of the of a analysis of the regulatory elements of sites with high and have in a of and of these are in locations in two or more of the four genes. the other three apoL-II has no All have at and is the large of sites many of which are containing a of four or of a of genes in lipid have sterol regulatory protein The apoL-I promoter has and apoL-II, apoL-III, and apoL-IV have putative In of the four genes are present in the sequences, which are found at or the transcription start in TATA-less S.T. from TATA-less within Biophys. Acta. 1997; Scholar), are present in in a in apoL-IV that the used promoter regions of the four apoL genes are TATA-less with transcription analysis of regulatory elements of the apoL gene high to the are The numbers to the to the in the in in in in between the four promoter in a new high to the are The numbers to the to the in the in in in in between the four promoter The of of apoL-III was is a sequence at from which was the start of our 5′ RACE is at and is a at The of the alternative apoL-II and apoL-III is is that are that the apoL family of genes in by TATA-less promoters, a associated with genes and Human on and Scholar). genes are expressed and Human on and Scholar), as is the with the apoL gene the from a poly A+ dot with an can be that apoL-I is expressed in a of the clones of apoL-I and apoL-III by RT-PCR The of these was examined in a of was to a PCR primer for gene in to the high We to from apoL-I, apoL-II, and apoL-III, using primer which is an for of these three a primer in the of gene. the of an RT-PCR using these with the of of the apoL-I apoL-II, and The apoL-I containing 2 is using this for apoL-II was The of the four apoL-III with the four found by sequencing The to exons and to a predicted to be to exons to exons and to exons The was more that with The with was to that found with liver, prostate, and in these four tissues the were The of the apoL-III is in the The GenBank accession numbers of the four common apoL-III are The two alternative of apoL-I to two proteins that in the of peptide The more common that 2 to The variant with To the total of mRNA for gene in a of we to primers and determine the for apoL-I, apoL-II, and apoL-III, of the homology between these three genes. The primers were designed to the in the the of all splice this we for apoL-IV, the primers used mRNA from this gene. a of type RT-PCR that the conditions for the quantitative competitive The of was determined to be from the RT-PCR control The RT-PCR that apoL-I is expressed at its highest in is apoL-II and that the in are of total the dot assay which more and is as of RNA was for the expression of eight genes. is the for the of expression of factor may be of the apoL-I used in the dot with other apoL gene In with analysis in our pancreatic expression of apoL-I was high The discovery of three additional genes for of the apoL family a family of four closely on chromosome that have an high of The presence of splice for the of gene products in this is of with a total of the 38-kDa variant of apoL-I that is found in plasma (9Duchateau P.N. Pullinger C.R. Orellana R.E. Kunitake S.T. Naya-Vigne J. O'Connor P.M. Malloy M.J. Kane J.P. Apolipoprotein L, a new human high density lipoprotein apolipoprotein expressed by the pancreas. Identification, cloning, characterization, and plasma distribution of apolipoprotein L.J. Biol. Chem. 1997; 272: 25576-25582Google Scholar) is the result of splice is The that 5′ sequences are in the transcription of the as to may on the of of for apoL-I may have the of apoL-IV, alternative a form with a peptide and suggesting that the gene in and has described for S. S. P. Tall A.R. of the mRNA the human transfer 1992; Scholar, C. Tall of the of transfer protein by a variant protein by alternative of Biol. Chem. Scholar). the apoL-III a protein with a The presence of amphipathic in all of the predicted gene products that could be in with in the of the selected and with plasma In the of the gene apoL-II, apoL-III, and that are to within roles in the of in the or of to may The high transcription for three of the of this family in that could have roles in the transport and in of the The presence of putative sterol elements in all four genes roles related to lipid synthesis or (10Duchateau P.N. Movsesyan I. Schoenhaus S.A. Spencer S.J. Mark S. Jaffe R.B. Redberg R.F. Kane J.P. Malloy M.J. Plasma apolipoprotein L levels are influenced by plasma triglyceride levels in normolipidemic, hyperlipidemic and NIDDM subjects.J. Lipid Res. 2000; 41: 1231-1236Google Scholar) that the of apoL-I in plasma is related to the of triglyceride a in the or transport of The that the transcription in for at of the of this family is increased or more to H. are to in are expressed in of and two novel Scholar) that this protein may have a in the could the of the that we have found in apoL-III gene. is to be present in HDL at a concentration to other HDL apolipoproteins. structure that could as a for lipid The presence of in predicted gene products of two other genes in this family at under in in with The of a of apoL-III to by may an of the lipoprotein to Funk C. for lipoproteins in from Scholar). this and in lipid transport or may roles for these proteins and in involving was by of by the by a from the and by from and and