Human protein NEFA, a novel DNA binding/EF-hand/leucine zipper protein. Molecular cloning and sequence analysis of the cDNA, isolation and characterization of the protein.

The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

The covalent linkage of secretory component to IgA. Structure of sIgA.

Immunoglobulin A which is secreted into external fluids is synthesized in plasma cells as an (IgA)2-J-chain complex. This complex docks on to the polyimmunoglobulin receptor which is located at the basolateral surface of epithelial cells. After docking the (IgA)2-J-receptor complex is internalized and processed. The polyimmunoglobulin receptor loses its C-terminal tail and thus becomes the secretory component. This secretory component is then covalently linked to the (IgA)2-J-chain complex by a disulfide bond, and protects the so formed sIgA from denaturation and proteolysis in external fluids. In order to establish this disulfide bond between IgA and the secretory component, sIgA, purified from human colostrum, was subjected to several enzymatic and chemical fragmentation reactions. One of the resulting polypeptides allowed us to characterize the covalent linkage of the secretory component to IgA in sIgA. IgA was found to be covalently linked to the secretory piece by a single disulfide bond between Cys 311 of one alpha-chain and Cys 467 of the secretory component. Cys 501 of the secretory component and Cys 311 of the other alpha-chain are blocked by cysteines. With this last paper of a series the structure of an entire sIgA molecule has been elucidated.

Intra- and interchain disulfide bridges of the human J chain in secretory immunoglobulin A.

All intra J chain disulfide bridges of human sIgA, the disulfide bonds between the J chain and the two IgA monomers, and one inter IgA monomer disulfide bridge were determined. sIgA was isolated from colostrum of healthy women and digested with IgA1-specific protease followed by cyanogen bromide cleavage. This procedure generated fragments of 140 kDa, 60 kDa, and 28 kDa. The 28-kDa polypeptide comprised the complete J chain covalently bound to two alpha 1 chain octapeptides derived from the C-termini of two alpha 1 chains. The 28-kDa fragment was digested with trypsin. The resulting peptides were purified by RP-HPLC, and subsequently characterized by amino-acid analysis, mass spectrometry, and gas phase sequencing. These data unequivocally show that the J chain cysteines C1-C6, C4-C5, and C7-C8 form intra chain disulfide bridges. The second (C2) and the third (C3) J chain cysteines are disulfide linked to two alpha chain cysteines (C17) joining the two IgA monomers of sIgA tail to tail. The remaining two alpha chains of the two monomers are directly bound to each other via their ultimate cysteines (C17-C17). A new model for the J chain in sIgA is presented.

Determination of the molecular structure of the human free secretory component.

Here, we present the experimental data, leading to determination of the primary structure, the linkage of the carbohydrates and the arrangements of the disulfide bonds of the human free secretory component. Methods of protein chemistry were used. The protein can be divided into five homology regions and is a member of the immunoglobulin superfamily.

[Purification and characterization of the free secretory piece of human colostrum (author's transl)]. / Reinigung und Charakterisierung der freien sekretorischen Komponente aus Human-Kolostrum.

The free secretory piece is isolated from human colostrum by gel filtration and ion-exchange chromatography in high yield (200 mg/l colostrum). DEAE-Cellulose chromatography separates the free secretory piece in two fractions which are electrophoretically distinct, but otherwise have the same characteristics, like molecular weight, antigenic determinants, N-terminal sequence, peptide map and amino acid composition. It was therefore concluded that the protein part of the secretory piece is homogenous.