Cloning of Ovocalyxin-36, a Novel Chicken Eggshell Protein Related to Lipopolysaccharide-binding Proteins, Bactericidal Permeability-increasing Proteins, and Plunc Family Proteins

The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20–25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens. The avian eggshell is a composite biomaterial composed of noncalcifying eggshell membranes and the overlying calcified shell matrix. The shell is deposited in a uterine fluid where the concentration of different protein species varies at different stages of its formation. The role of avian eggshell proteins during shell formation remains poorly understood, and we have sought to identify and characterize the individual components in order to gain insight into their function during elaboration of the eggshell. In this study, we have used direct sequencing, immunochemistry, expression screening, and EST data base mining to clone and characterize a 1995-bp full-length cDNA sequence corresponding to a novel chicken eggshell protein that we have named Ovocalyxin-36 (OCX-36). Ovocalyxin-36 protein was only detected in the regions of the oviduct where egg-shell formation takes place; uterine OCX-36 message was strongly up-regulated during eggshell calcification. OCX-36 localized to the calcified eggshell predominantly in the inner part of the shell, and to the shell membranes. BlastN data base searching indicates that there is no mammalian version of OCX-36; however, the protein sequence is 20–25% homologous to proteins associated with the innate immune response as follows: lipopolysaccharide-binding proteins, bactericidal permeability-increasing proteins, and Plunc family proteins. Moreover, the genomic organization of these proteins and OCX-36 appears to be highly conserved. These observations suggest that OCX-36 is a novel and specific chicken eggshell protein related to the superfamily of lipopolysaccharide-binding proteins/bactericidal permeability-increasing proteins and Plunc proteins. OCX-36 may therefore participate in natural defense mechanisms that keep the egg free of pathogens. The avian eggshell is a calcitic bioceramic, which possesses unique mechanical properties deriving from its complex a porous polycrystalline structure. It rests upon, and is continuous with, an underlying noncalcified meshwork of fibers. The eggshell membranes, the calcified (mammillary, cone, and palisade) layers and cuticle of the eggshell, are sequentially laid down upon the egg white in the distal part of the oviduct (isthmus and uterus). The eggshell mineral is associated with an organic matrix composed of proteins, glycoproteins, and proteoglycans, which are thought to influence the fabric of this biomaterial (1Nys Y. Gautron J. Garcia-Ruiz J.M. Hincke M.T. C. R. Palevol. 2004; 3: 549-562Crossref Scopus (366) Google Scholar). Matrix protein components have been identified in decalcified shell and in the uterine fluid bathing the egg during its formation, and this contains the organic and mineral precursors of the eggshell (2Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (117) Google Scholar). Previously identified matrix protein components can be divided into three characteristic groups as follows: (i) “egg white” proteins, which are also present in the eggshell, and these include ovalbumin (3Hincke M.T. Connect. Tissue Res. 1995; 31: 227-233Crossref PubMed Scopus (105) Google Scholar), lysozyme (4Hincke M.T. Gautron J. Panheleux M. Garcia-Ruiz J.M. McKee M.D. Nys Y. Matrix Biol. 2000; 19: 443-453Crossref PubMed Scopus (207) Google Scholar), and ovotransferrin (5Gautron J. Hincke M.T. Panhé;leux M. Garcia-Ruiz J.M. Boldicke T. Nys Y. Connect. Tissue Res. 2001; 42: 255-267Crossref PubMed Scopus (127) Google Scholar); (ii) ubiquitous proteins that are found in many tissues, and this group includes osteopontin, a phosphorylated glycoprotein present in bone and other hard tissues (6Pines M. Knopov V. Bar A. Matrix Biol. 1994; 14: 765-771Crossref Scopus (107) Google Scholar, 7Hincke M.T. St. Maurice M. Goldberg M. Boskey A. Robinson C. Chemistry and Biology of Mineralized Tissues: Proceedings of the Sixth International Conference. 2000: 13-17Google Scholar, 8Fernandez M.S. Escobar C. Lavelin I. Pines M. Arias J.L. J. Struct. Biol. 2003; 143: 171-180Crossref PubMed Scopus (56) Google Scholar), and clusterin, a widely distributed secretory glycoprotein that is also found in chicken egg white (9Mann K. Gautron J. Nys Y. McKee M.D. Bajari T. Schneider W.J. Hincke M.T. Matrix Biol. 2003; 22: 397-407Crossref PubMed Scopus (69) Google Scholar); and (iii) matrix proteins unique to the shell calcification process that are secreted by cells in specific regions of the oviduct where egg-shell mineralization is initiated and continues to and uterus). was the eggshell protein from the shell M.T. M. V. R. Tissue 1995; PubMed Scopus Google Scholar). It is a of K. Biol. Google that in and in the shell matrix K. PubMed Scopus Google Scholar). was the eggshell matrix protein to be M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). is the protein of a eggshell M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar, Connect. Tissue Res. 1997; 36: PubMed Scopus Google Scholar, M.S. A. Arias J.L. Matrix Biol. 2001; 19: PubMed Scopus Google Scholar), which is found the calcified eggshell M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). we a protein that is in the calcified and cuticle of the calcified shell J. Hincke M.T. K. Panhé;leux M. M. McKee M.D. Nys Y. J. Biol. 2001; PubMed Scopus Google Scholar, J. Hincke M.T. K. McKee M.D. M. Nys Y. I. Proceedings of the International Scholar, M.T. Gautron J. K. Panheleux M. McKee M.D. M. Nys Y. Connect. Tissue Res. 2003; PubMed Scopus Google Scholar). The natural of the egg have components as follows: is the shell that as a and the other is a composed of proteins that have been identified in egg white M. Nys Y. Conference. Scholar). It been that in eggshell properties are related to of egg and of K. J. Scopus Google Scholar). In we have that proteins with properties are associated with the is in the that the egg white and the of the shell membranes. It is also present in the shell membranes and in the matrix and cuticle of the calcified shell (4Hincke M.T. Gautron J. Panheleux M. Garcia-Ruiz J.M. McKee M.D. Nys Y. Matrix Biol. 2000; 19: 443-453Crossref PubMed Scopus (207) Google Scholar). is localized in the calcified and in the eggshell membranes, where as a to of in egg white (5Gautron J. Hincke M.T. Panhé;leux M. Garcia-Ruiz J.M. Boldicke T. Nys Y. Connect. Tissue Res. 2001; 42: 255-267Crossref PubMed Scopus (127) Google Scholar). In this study, we the cDNA and protein genomic organization and of expression of Ovocalyxin-36 a novel protein that is homologous to of mammalian protein protein is the of an protein that may participate in the natural defense of the egg of and to a of of by of a as by the Scholar). was with a of the of fluid was as (2Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (117) Google Scholar). was by of of at and of the egg with this These to the and of shell fluid was egg into a at the of the of uterine fluid with and Tissue and of the oviduct white and and and other and from an egg was in the of the during the of calcification uterine tissues of at related to and egg formation from the (i) with a oviduct the of egg (ii) in which of was during to the egg mineralization and (iii) in which the was during eggshell calcification. and of Matrix matrix proteins as (5Gautron J. Hincke M.T. Panhé;leux M. Garcia-Ruiz J.M. Boldicke T. Nys Y. Connect. Tissue Res. 2001; 42: 255-267Crossref PubMed Scopus (127) Google with The with and in used the of shell to the of shell membranes from the inner part of the calcified (i) (ii) egg was with by the mechanical of the membranes The egg was with the (iii) with and the membranes The with with and the membranes The with these to associated shell membranes, to a decalcified at in The was and was at with in the was of and The of matrix proteins these are to as egg-shell protein with uterine fluid at the of calcification of the with and with The was and into a with a The was in with and into The at with the in to a an used lipopolysaccharide-binding bactericidal permeability-increasing was a corresponding to a sequence the of the OCX-36 protein The was to and used of the the was with with in was at and and the and protein concentration of was by the PubMed Scopus Google ovalbumin as the and a as (2Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (117) Google Scholar, J. Hincke M.T. K. Panhé;leux M. M. McKee M.D. Nys Y. J. Biol. 2001; PubMed Scopus Google Scholar). fluid and eggshell in and and to the with the (i) in (ii) in membranes in and in The membranes with in in the membranes with in The membranes in and in The membranes an in the are the in this and of the protein at the was by and membranes. The membranes with and a and in the The was into a protein and to by in an protein the was to by The by and into the protein uterine fluid at the was by The corresponding to the was with a into The and and with and at in a with in as A. M. M. PubMed Scopus Google Scholar). The with and by The matrix in and the the the The in with an of V. data the was the from of was data by to a with a and to be as precursors a of The was the and The was the of in The a and with at by a with a at a of The and to proteins in a data base the cDNA cDNA was from uterine during the of eggshell calcification as M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). was with The was to the uterine fluid protein and as and detected with and with to by three of and into was by and the cDNA by at the of clone with a novel of in was by expression this sequence and used to the expression of this message in different of the oviduct and in was only detected in the uterine of the The was and used of the to In this and by at the of the of of of the full-length of of used in a and by was from a and with by at and the of was a of and was to and of the OCX-36 the and at and OCX-36 cDNA was in the with the in and OCX-36 to The a and as (9Mann K. Gautron J. Nys Y. McKee M.D. Bajari T. Schneider W.J. Hincke M.T. Matrix Biol. 2003; 22: 397-407Crossref PubMed Scopus (69) Google Scholar). specific from the OCX-36 cDNA sequence The of and of the of and The of a at by a at and of at and at was from to in individual with a with a specific of the of and of the of was and in in the of no and of cDNA corresponding to a of uterine cDNA from during eggshell formation. The cDNA was from to and The as the at which a base was and was the of the cDNA and of from this The and and The was as The of the was used version of was to in OCX-36 expression in at was the M. A. M. C. K. V. M. A. PubMed Google Scholar). The and the was in of of of of and at at at and at by a of at a and was used as and and as of OCX-36 in and of oviduct tissues and decalcified eggshell and as (3Hincke M.T. Connect. Tissue Res. 1995; 31: 227-233Crossref PubMed Scopus (105) Google Scholar, M.T. M. V. R. Tissue 1995; PubMed Scopus Google Scholar). In tissues from in in and in and in of shell and decalcified at in a of and in decalcified to to the the shell in The was at and as in an with a by the to the of the egg in and an into and with to the OCX-36 at in at In as the was in and at with the was by in and in in in and in as M.D. A. Res. 1995; 31: PubMed Scopus Google Scholar). of by and regions and in by with and detected by a protein complex of The with and and in a at of the of from to eggshell in and with to an from the inner of the from the egg white with a and was a at an of cDNA and of fluid the egg during its formation and contains eggshell matrix components as precursors (2Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (117) Google Scholar). protein varies during of the three stages of the calcification process by during the is a protein and membranes, this was to to the sequence of the protein three to be and was with the sequence to the sequence of the of the uterine fluid protein the of the uterine fluid The was from the and into to that used to an expression cDNA with from uterine during the calcification and to the protein of a of eggshell matrix M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar), and three to that we have as a uterine fluid protein (9Mann K. Gautron J. Nys Y. McKee M.D. Bajari T. Schneider W.J. Hincke M.T. Matrix Biol. 2003; 22: 397-407Crossref PubMed Scopus (69) Google Scholar). clone a novel cDNA sequence of the used the of the OCX-36 full-length of to the sequence are by of sequence is by and to and to of the and by the at the of the that from the novel and the was of the uterine to and to a composite cDNA sequence BlastN the composite sequence identified and and and from the of cDNA in by EST from the chicken cDNA by the of Nys Y. J. R. J. J. PubMed Scopus Google Scholar). EST clone from the cDNA in J. C. Biol. PubMed Scopus Google Scholar). The and and and with the of the of in the a 1995-bp full-length cDNA sequence named the corresponding protein Ovocalyxin-36 BlastN of the chicken sequence version of was The OCX-36 was and The OCX-36 genomic sequence is and of in from to with that in from to was by with the The to the and the identified by to the The was to OCX-36 with a of and at a of The of OCX-36 the can be the organization of the OCX-36 in to are to the cDNA sequence at the are to the cDNA sequence at the in a and and genomic used to a cDNA sequence of the cDNA the is at and the at and The the the and the base of the The is in of the the and the The protein is in corresponding to a The of the at with a three is with the sequence of in species M. J. Biol. PubMed Scopus Google Scholar, M. PubMed Scopus Google Scholar). The is and of the the sequence to the by direct of the uterine fluid protein The protein is therefore to be corresponding to a of The a of and and the is The by direct of the are found in the sequence from the cDNA and to and of the OCX-36 the was by and the of that to the from the cDNA sequence from and identified by in the have these to be eggshell matrix proteins, and we detected and in this by (9Mann K. Gautron J. Nys Y. McKee M.D. Bajari T. Schneider W.J. Hincke M.T. Matrix Biol. 2003; 22: 397-407Crossref PubMed Scopus (69) Google Scholar, M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). The of OCX-36 that direct to the and that the and regions of the and and and of the cDNA to the cDNA sequence regions where no sequence was These used in with from chicken uterine The to the of the cDNA with that to be in the in a the OCX-36 composite cDNA sequence by of EST The and are the of and of the clone from cDNA These in of at of the cDNA sequence of the a a is in of at the of the the of the sequence remains The EST from the cDNA and a at and of the sequence and of the The in a sequence of the of the which to a at by an The sequence is of Ovocalyxin-36 BlastN the data base other sequence with In with mammalian proteins bactericidal permeability-increasing proteins and proteins and with and clone family proteins in and The sequence is 20–25% to and to Plunc family proteins. The from to The OCX-36 and these proteins from to a these proteins. The OCX-36 with and proteins is by data base searching protein and with the from the and the in proteins. Ovocalyxin-36 protein with the family with from to The of the of the OCX-36 and of the OCX-36 sequence to the and of the family proteins. a was detected at the in proteins, this was detected by direct of this by protein and by and detected by of the of this of OCX-36 sequence was the of and of distributed the sequence of Ovocalyxin-36 in the to a from the sequence was used and The of OCX-36 the uterine fluid and in eggshell was by strongly was in the uterine fluid during the calcification was also in the and of shell calcification in uterine fluid and of also in uterine fluid during the where the was the which to of the to of OCX-36 as M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). of the in the of the The that Ovocalyxin-36 is an eggshell matrix protein was by eggshell in which eggshell membranes from shell as follows: mechanical only and of the membranes in the in which the of the shell was with the the of the a of of OCX-36 in the shell was from the where no The of a mechanical and of membranes that OCX-36 is an eggshell matrix protein eggshell OCX-36 is present in from shell with membranes and and with membranes and in only calcified part of the shell and no membranes The and of OCX-36 decalcified was by in order to the was the eggshell and in the membranes The was the at the the membranes and the calcified where mineralization of the shell is at the shell was as OCX-36 by with by of the egg-shell These observations and to was in the shell membranes and in the calcified part of the shell, where was in the In the inner membranes to the egg OCX-36 was present in the and the of the in the membranes with the of the shell, the was and only the Tissue and of of OCX-36 was The expression of OCX-36 was in of the oviduct in the of egg components and in other OCX-36 expression was only detected in oviduct regions where eggshell calcification takes and expression was detected in the oviduct and white in other and of expression was The of OCX-36 message to to in and The OCX-36 expression was in the and different from the other tissues In the the expression was that in the and was that detected in other tissues where the OCX-36 expression was The was also used to the expression of OCX-36 in from with a oviduct to and egg group was with from in which the egg was eggshell to the in expression by and associated In OCX-36 uterine expression was that there was a egg in the group of the by of the egg by the process of eggshell calcification in of The expression of was in the expression of OCX-36 to a that in the OCX-36 uterine expression was an egg was present in the during eggshell calcification of OCX-36 an egg the and was shell calcification was by with uterine expression at the of egg white the egg is in the OCX-36 expression was in of with a calcified egg eggshell with no shell was and the egg was in the of the oviduct The of OCX-36 protein in other tissues was by The was detected in tissues however, its in white and was in tissues at uterine was at The was detected in and in egg white at was detected in and was in the of OCX-36 was by of uterine a of the cells in a of was the of the which to of secreted The eggshell is a that is in avian It the of and the and the It as a mechanical to the egg from the and the and of the eggshell are of the In this we the of a novel protein with an as by of protein was identified in as a by (2Gautron J. Hincke M.T. Nys Y. Connect. Tissue Res. 1997; 36: 195-210Crossref PubMed Scopus (117) Google and was found to be in uterine fluid during the of calcification. OCX-36 was used to a cDNA expression with from uterine clone was and used The sequence was with to a full-length The cDNA and corresponding protein sequence are and was therefore named Ovocalyxin-36 shell, and its The from this cDNA to a protein of The regions of the protein that direct to and that and regions of the the the of the uterine fluid protein and that of the be by a of the no and mechanisms as this this we to in the the OCX-36 sequence the that the protein sequence which no be that are in the cDNA was we that OCX-36 is as a full-length with no and that full-length protein with the to the also identified with and of of the a specific to OCX-36 was a with this a corresponding in uterine fluid during the calcification and of shell formation protein was in uterine fluid during the calcification where a of was this was to in the of we that OCX-36 is to as that by in the white of the oviduct PubMed Scopus Google Scholar). of OCX-36 of is to its highly as been V. R. R. A. PubMed Scopus Google Scholar). in of an the the sequence and that detected by as in the to OCX-36 protein to of the OCX-36 its in decalcified eggshell that into the calcified was by to shell with no organic fibers. the eggshell membranes only with organic the calcified part of the shell the shell was with by and the membranes be from the calcified part of the shell OCX-36 was in with and and membranes, that OCX-36 is also a matrix protein of the calcified was by at the and where OCX-36 was detected the eggshell, with the in the inner of the shell to the membranes of Ovocalyxin-36 is in with the of the organic matrix of the chicken eggshell in which OCX-36 was identified as a protein of the calcified matrix K. PubMed Scopus Google Scholar). other proteins have been localized in the of the eggshell. is in the shell J. Hincke M.T. K. Panhé;leux M. M. McKee M.D. Nys Y. J. Biol. 2001; PubMed Scopus Google Scholar), is detected in the M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar). the other is distributed the and the M.T. M. V. R. Tissue 1995; PubMed Scopus Google Scholar). The expression of OCX-36 was and OCX-36 message was only in the where egg-shell calcification takes expression be detected in the other of the oviduct in other OCX-36 protein was detected in white and that of OCX-36 protein the of the oviduct from its distal and the white OCX-36 in this its with the shell membranes. of secretory uterine of the and of the underlying are in of eggshell matrix proteins. The and are secreted by the M.T. Gautron J. McKee M.D. Nys Y. J. Biol. PubMed Scopus Google Scholar, J. Hincke M.T. K. Panhé;leux M. M. McKee M.D. Nys Y. J. Biol. 2001; PubMed Scopus Google Scholar). In OCX-36 is secreted by cells as and (5Gautron J. Hincke M.T. Panhé;leux M. Garcia-Ruiz J.M. Boldicke T. Nys Y. Connect. Tissue Res. 2001; 42: 255-267Crossref PubMed Scopus (127) Google Scholar, K. Gautron J. Nys Y. McKee M.D. Bajari T. Schneider W.J. Hincke M.T. Matrix Biol. 2003; 22: 397-407Crossref PubMed Scopus (69) Google Scholar, M.T. M. V. R. Tissue 1995; PubMed Scopus Google Scholar). of OCX-36 expression during egg formation was by expression OCX-36 was highly in the of with a eggshell, with expression of the egg and of calcification. the expression of into the is up-regulated by the of the egg into the I. Bar A. Pines M. Matrix Biol. PubMed Scopus Google Scholar). expression is to the mechanical upon the uterine expression of a and is up-regulated by the mechanisms McKee PubMed Scopus Google Scholar, I. R. Pines M. J. 2001; Scholar, I. M. Pines M. J. Scholar). an role OCX-36 protein its protein sequence with and Plunc family proteins. These proteins are in their in defense to the superfamily of proteins to be components of the innate immune which as the of defense 2004; PubMed Scopus Google Scholar). OCX-36 and the family of mammalian proteins is by of their structure. The organization of the OCX-36 is to that of the highly and C. C. Res. 1997; PubMed Scopus Google Scholar), with corresponding OCX-36 to which a of with the of and These data strongly suggest a and with a in the mammalian and to the at the OCX-36 and the is composed of and J. J. Biol. PubMed Google Scholar). the in proteins, we that and of the OCX-36 protein sequence the and of proteins, OCX-36 is to and protein as in the 1997; PubMed Google Scholar). chicken with was by to be a egg white C. M. C. T. J. PubMed Scopus Google Scholar). sequence that and OCX-36 have only with other to OCX-36 and are therefore different egg proteins with sequence to proteins. we that OCX-36 to natural egg by the egg in the of the distal oviduct during its formation in the white and the chicken eggshell is a the egg be present in the from the eggshell formation. an natural to of the egg is a function that been to egg white proteins that properties M. Nys Y. Conference. Scholar). the in of by which is their have that these components of egg white are also eggshell matrix proteins (4Hincke M.T. Gautron J. Panheleux M. Garcia-Ruiz J.M. McKee M.D. Nys Y. Matrix Biol. 2000; 19: 443-453Crossref PubMed Scopus (207) Google Scholar, J. Hincke M.T. Panhé;leux M. Garcia-Ruiz J.M. Boldicke T. Nys Y. Connect. Tissue Res. 2001; 42: 255-267Crossref PubMed Scopus (127) Google and may also influence the calcification also function as in the eggshell and its membranes. Moreover, eggshell matrix and which be by the of lysozyme ovotransferrin Y. C. J. 2003; PubMed Scopus Google Scholar). may a with proteins as In this we have that OCX-36 is in the shell and associated membranes. from the at the of the to in response to secreted by the process OCX-36 and as the eggshell in In OCX-36 is a novel eggshell protein that is secreted during shell formation and is found in the of the distal into the eggshell membranes and the eggshell. OCX-36 may be in the innate defense of the egg to keep the egg free of and the the are to M. and to A. at and to the of the and also M. and A. their and J. the of The of and is

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