Molecular cloning and ultrastructural localization of the core protein of an eggshell matrix proteoglycan, ovocleidin-116

The role of avian eggshell matrix proteins in shell formation is poorly und erstood. This calcitic biomaterial forms in a uterine fluid where the prote in composition varies during the initial, calcification, and terminal phase s of eggshell deposition, A specific antibody was raised to a 116-kDa prote in, which is most abundant in uterine fluid during active eggshell calcific ation. This antiserum was used to expression screen a bacteriophage cDNA li brary prepared using mRNA extracted from pooled uterine tissue harvested at the midpoint of eggshell calcification. Plasmids containing inserts of dif fering 5'-lengths were isolated with a maximum cDNA sequence of 2.4 kilobas es, Northern blotting and reverse transcriptase-polymerase chain reaction d emonstrated that the 2,35-kilobase message was expressed in a uterine-speci fic manner,. The hypothetical translational product from the open reading f rame corresponded to a novel 80-kDa protein, which we have named ovocleidin -116, After removal of the predicted signal peptide, its N-terminal sequenc e corresponded almost exactly with that determined from direct microsequenc ing of the 116-kDa uterine protein (this work) and with that previously det ermined for the core protein of a 120-kDa egg shell dermatan sulfate proteo glycan (Corrino, D. A., Rodriguez, J, P,, and Caplan, A. I. (1997) Connect. Tissue Res. 36, 175-193), Ultrastructural colloidal gold immunocytochemist ry of ovocleidin-116 demonstrated its presence in the organic matrix, in sm all vesicles found throughout the mineralized palisade layer, and the calci um reserve assembly of the mammillary layer. Ovocleidin-116 thus is a candi date molecule for the regulation of calcite growth during eggshell calcific ation.