EMILIN, a Component of the Elastic Fiber and a New Member of the C1q/Tumor Necrosis Factor Superfamily of Proteins

EMILIN (elastinmicrofibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil α helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of β-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative. EMILIN (elastinmicrofibril interface located protein) is an extracellular matrix glycoprotein abundantly expressed in elastin-rich tissues such as blood vessels, skin, heart, and lung. It occurs associated with elastic fibers at the interface between amorphous elastin and microfibrils. Avian EMILIN was extracted from 19-day-old embryonic chick aortas and associated blood vessels and purified by ion-exchange chromatography and gel filtration. Tryptic peptides were generated from EMILIN and sequenced, and degenerate inosine-containing oligonucleotide primers were designed from some peptides. A set of primers allowed the amplification of a 360-base pair reverse transcription polymerase chain reaction product from chick aorta mRNA. A probe based on a human homologue selected by comparison of the chick sequence with EST data base was used to select overlapping clones from both human aorta and kidney cDNA libraries. Here we present the cDNA sequence of the entire coding region of human EMILIN encompassing an open reading frame of 1016 amino acid residues. There was a high degree of homology (76% identity and 88% similarity) between the chick C terminus and the human sequence as well as between the N terminus of the mature chick protein where 10 of 12 residues, as determined by N-terminal sequencing, were identical or similar to the deduced N terminus of human EMILIN. The domain organization of human EMILIN includes a C1q-like globular domain at the C terminus, a collagenous stalk, and a longer segment in which at least four heptad repeats and a leucine zipper can be identified with a high potential for forming coiled-coil α helices. At the N terminus there is a cysteine-rich sequence stretch similar to a region of multimerin, a platelet and endothelial cell component, containing a partial epidermal growth factor-like motif. The native state of the recombinantly expressed EMILIN C1q-like domain to be used in cell adhesion was determined by CD spectra analysis, which indicated a high value of β-sheet conformation. The EMILIN C1q-like domain promoted a high cell adhesion of the leiomyosarcoma cell line SK-UT-1, whereas the fibrosarcoma cell line HT1080 was negative. Elastic fibers are major constituents of the extracellular matrix (ECM), 1The abbreviations used are: ECM, extracellular matrix; gC1q-like, globular C1q-like domain; TNF, tumor necrosis factor; PCR, polymerase chain reaction; EST, Expressed Sequence Tag; bp, base pair(s); RACE, rapid amplification of cDNA ends; CAFCA, centrifugal assay for fluorescence-based cell adhesion confer to connective tissues the properties of resilience and elastic recoil, and are remarkable for the diverse range of tissues in which they are found. Elastic fibers can be identified in the ECM of many tissues as solid branching and unbranching fine and thick rod-like fibers (in elastic ligaments) or as concentric sheets of lamellae (in blood vessels) or in three-dimensional meshworks of fine fibrils (in elastic cartilage) or as a combination of these (in skin and lung) (1Partridge S.M. Adv. Protein Chem. 1962; 17: 227-302Crossref Scopus (126) Google Scholar). 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Full Text PDF PubMed Google Scholar). that EMILIN is expressed in connective tissues and is in blood vessels, skin, heart, and whereas is in the A. J. Cell Biol. PubMed Scopus Google Scholar, A. PubMed Scopus (11) Google Scholar, A. A. A. 7: PubMed Scopus Google the protein is by and by and is as a fine A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. P. J. Biol. Chem. Full Text PDF PubMed Google EMILIN by to high A. P. J. Biol. Chem. Full Text PDF PubMed Google EMILIN is a component of elastic fibers and is at the interface between amorphous elastin and A. J. Cell Biol. 1993; PubMed Scopus (128) Google and for the of the of elastin in is by the of in the A. J. Cell Biol. 1993; PubMed Scopus (128) Google Scholar). the of elastin and the fine at the interface between elastin and and the with the of elastin in is that EMILIN a in the of also in the of the of EMILIN we to the cDNA sequence and the of the deduced amino acid sequence of human EMILIN. a of the of proteins, is by a a collagenous two leucine and an coiled-coil region that is with of this C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). At the N terminus of these two of the there is a region of homology including a partial epidermal growth factor-like motif. the recombinantly EMILIN domain is to cell EMILIN was purified as A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). aortas and associated blood vessels were from 19-day-old chick and into containing the 2 and 1 were at 4 The tissues were and the at for was in the and in containing and The was extracted in containing The two were and The was and and was by chromatography by gel filtration. The as by an assay and by with a specific A. PubMed Scopus (11) Google Scholar), were and of the was by gel to for the of EMILIN was on a The to EMILIN was into and with by was The gel were for 2 and by of containing at The 1 of gel and as was used as was to the gel was at and the peptides were extracted with acid and with acid in the gel were with and and was with a at and The peptides were by high chromatography on a a of in Sequence was on a protein to the The amino acid were used to the protein sequence data base and of of the chick EMILIN peptides for the of degenerate primers A set of degenerate inosine-containing based on peptides and were on an in both the and the The primers were purified by native gel a from aortas of 19-day-old chick was The was purified from with the of The cDNA was from 1 of with and with of was on a with the degenerate inosine-containing used as primers in the and at from to with 2 The amplification were into the and by the chain the polymerase the human EMILIN the W.R. U. S. A. PubMed Scopus Google Scholar) located at the W. M. J. Mol. Biol. 1990; PubMed Scopus Google Scholar) was used to the data base for expressed sequence containing to of chick EMILIN. human and a homology to the of the chick EMILIN. and were designed based on the and used in to a human The reaction was on a by kidney with and with of reverse The amplification product was into the and to The was by the with the and to by the clones of a human kidney cDNA in the Inc., The of the was used to the The by a between a specific EMILIN cDNA and an additional of of a human aorta cDNA in the of clones and in this several clones to be to The human were the and a for polymerase were determined from both and were on clones from human cDNA were by the EMILIN by an of a used to the EMILIN A. F. M. P. and A. in the sequence the the the was of chick aorta was with the of and the product was to the EMILIN specific in combination with the from the The EMILIN domain was by from in the and in for generated by PCR, the were in both were at for and the cell was in at of The was in a in and on and cell was by the of at A The cell was at for the was and of the EMILIN C1q-like domain was by chromatography on acid native The protein was from the in containing and the C1q-like domain was used for CD spectra and cell adhesion The purified EMILIN was used for CD A to an for data was used for the of the was with acid at were 10 a The was by a spectra are as of The are the 10 the data were in of based on a of the spectra were the L. J. M. A. F. 4: Scholar). The of human EMILIN domain to cell was several cell and the centrifugal assay for fluorescence-based cell adhesion E. J. A. R. (in Google Scholar, P. E. R. in Inc., Scholar). of Inc., with were with EMILIN and with for at to of the human and and a human cell were by with the The in and and into the at a of Cell adhesion to was in containing 1 and and as a were on and at for at to of the with the The were for at and with a similar lacking such as to to be The of to the to the and (in of the was by in a were by to the of the cell to a E. J. A. R. (in Google Scholar, P. E. R. in Inc., Scholar). A chick aorta EMILIN and chromatography was by gel and to be composed of a major with an of was in with A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the of the as by of the gel The protein was by and by a the peptides were by reverse chromatography on a and several were selected for on the several degenerate in the and were and used to from of chick amplification of were the of The sequence of the that but in the the the amplification product with the of the primers to peptides and an open reading frame that that the sequence to that of EMILIN protein the of the for to of of chick overlapping cDNA clones were from human kidney and aorta a human specific probe based on the EST and from at the of the of the human cDNA was by the high of clones from both libraries. The partial chick cDNA to an open reading frame of amino The coding sequence and the deduced amino acid sequence of the human EMILIN cDNA is in overlapping EST an with the of the human EMILIN including the of the coding sequence and the entire and also of clones the present The open reading frame of the human EMILIN with a into the M. PubMed Scopus Google Scholar) and is by an The human cDNA and has an open reading frame of 1016 amino The with the for the of the mature protein are between 2 value of and value of of the present the for the of the is at between the N-terminal sequence of the chick mature EMILIN and the deduced N terminus of the human EMILIN the 1 as the for the of the mature to a the sequence well with the sequence H. J. S. G. Protein PubMed Scopus Google Scholar) and with a J. Mol. Biol. PubMed Scopus Google Scholar). the for the mature protein is The human EMILIN and with a of as by or two that be in the additional to the sequence be The that the EST the of the present with a potential sequence the of that product includes the entire The of the stretch of chick EMILIN and the region of human EMILIN a high degree of homology the degree of amino acid sequence identity and in this domain was to be and a partial sequence of the N terminus of chick EMILIN well are and 2 are with the deduced N terminus of the human EMILIN The high of and between chick and human EMILIN at both C and N that the two the protein in these The domain structure of human EMILIN is from the of a globular domain an stretch of that EMILIN a stalk, and a amino acid sequence in which there are several heptad repeats by with the potential for forming coiled-coil A sequence that includes two to structures to as the which are of several proteins PubMed Scopus Google Scholar, P. 1990; 6: Full Text PDF PubMed Scopus Google Scholar), is located between the coiled-coil region and the collagenous is for an ECM as there are a in the for the of leucine in the ECM protein J. 1996; PubMed Scopus Google Scholar) and the protein leucine zipper has to with Caskey C.T. 1994; PubMed Scopus Google Scholar). the leucine zipper is from is at the be the of in the of EMILIN. a of the data indicated the of a region of homology between the N-terminal of EMILIN and the platelet and endothelial specific C. 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Cell adhesion to was high and for cell with a cell of the EMILIN domain promoted a high of adhesion of whereas HT1080 were and with a adhesion to a cell cell adhesion to the EMILIN domain was by a of cell The were for the with or with a of a that cell adhesion to the EMILIN domain adhesion to and is by that the cell adhesion of and used to assay adhesion to are to cell adhesion to the C1q-like domain of adhesion of and and HT1080 cell Cell adhesion to human and and to the human EMILIN domain purified native The proteins were at 10 and the cell adhesion assay was in containing 1 and 1 to with was microscopy of and are The provided in this a human cDNA major were a a collagenous and an domain containing with the potential of forming coiled-coil The of the structure cDNA and the of this sequence to human EMILIN was by the identity at the C terminus between the chick EMILIN sequence and that of the human the that the deduced sequence of of chick EMILIN 1 of the EMILIN that the sequence from chick aorta to that of the EMILIN by A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, A. J. Cell Biol. 1993; PubMed Scopus (128) Google Scholar, A. J. Cell Biol. PubMed Scopus Google Scholar, A. PubMed Scopus (11) Google Scholar, A. A. A. 7: PubMed Scopus Google Scholar, A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) as an protein with the sequence at the the identity between the N-terminal of the mature chick and the deduced amino of the mature human protein the that we are with the ECM in the two that chick EMILIN with A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and that in a with the apparent of A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar) indicated that chick EMILIN was highly The present of in the human EMILIN sequence is in with the data the chick A. J. Biol. Chem. Full Text PDF PubMed Google A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of with a high potential for is also in with the that and chick EMILIN as a in but was present as a that the gel in the of A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The domain organization of EMILIN is with several of the and such as the domain and a collagenous but also EMILIN an and coiled-coil region that is in the of the multimerin, a glycoprotein in and endothelial of P. PubMed Google Scholar) and with factor which is with in the E. J.C. G. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). There is that several of the to that are expressed as proteins or such as in Scholar, M. M. J. 1995; Google Scholar) or to as in S. F. E. J. U. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and E. P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). EMILIN is also to similar a which is highly to of the of the and an collagenous which can a the of the and an collagenous The EMILIN with the has a longer of a the both the and of proteins and in the of the core of the L. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar) are present in EMILIN domain in the and this to a similar and organization also for the domain of EMILIN. The potential homology between the of growth and the of the of proteins that these diverse from with L. Biol. 1998; Full Text Full Text PDF PubMed Google Scholar). for the proteins containing are cell these are well in TNF, but data are also for the component L. J. 1994; PubMed Scopus Google Scholar, J. 1994; Google Scholar, M. J. 1996; Google Scholar). several cell are with the to to the component cell two of structures a protein that the collagenous domain R. J. Biol. 1993; PubMed Scopus Google Scholar) and component that to the domain L. J. 1994; PubMed Scopus Google Scholar). the of this of has J. W. M. W. J. 1998; Google Scholar), and are The that EMILIN domain cell for some but to be for is with the and that cell of this domain be specific cell The adhesion was high for within the frame of the cell adhesion was by a is that from such as by are the of the adhesion is this adhesion a or an between amorphous elastin and in the aorta of Histochem. J. PubMed Scopus Google Scholar), and the of EMILIN A. J. Cell Biol. 1993; PubMed Scopus (128) Google Scholar) that the between the elastin amorphous core and the also an EMILIN EMILIN was in of aorta in with a of fibrils A. J. Cell Biol. 1993; PubMed Scopus (128) Google Scholar), EMILIN can be an in and this is by the that the of elastic in was by the of EMILIN A. J. Cell Biol. 1993; PubMed Scopus (128) Google Scholar). the of elastin and elastic is cell adhesion the EMILIN domain to be P. PubMed Google Scholar), is and can be as in the of aorta A. P. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The to coiled-coil potential to into of the heptad has a to and in two the is that these heptad repeats can is the are high that the can from the C1q-like domain at the C terminus, the collagenous domain to as in S. F. E. J. U. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and E. P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). are to EMILIN are into the the EMILIN domain is a for and the heptad repeats into or can also with EMILIN these the of and EMILIN in is in R. and A. and for for in the cell adhesion and for the CD spectra