NUCB1, the Drosophila melanogaster homolog of the mammalian EF-hand proteins NEFA and nucleobindin.

Mammalian NEFA and nucleobindin are calcium-binding proteins containing a signal peptide, two EF-hand motifs, acidic and basic regions and a leucine-zipper motif. Although they have been discussed to be involved in autoimmunity, apoptosis and calcium homeostasis in the Golgi apparatus and bone matrix, their exact role remains unknown. Here we report the cloning of their Drosophila homolog, nucb1, as well as the analysis of its expression pattern during embryogenesis and the subcellular localization of the NUCB1 protein. The nucb1 mRNA and the NUCB1 protein were found to be expressed maternally and zygotically, and they accumulate ubiquitously at low levels during all embryonic stages due to a maternal component. From stage 11 onward, high levels of zygotic expression can be detected specifically in the salivary glands and their placodes. In contrast to the known mammalian family members, the NUCB1 protein localizes in a subpattern of cytoplasmic substructures, probably the Golgi apparatus.

Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes.

Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.

The plasma membrane of Xenopus laevis oocytes contains voltage-dependent anion-selective porin channels.

Recent patch-clamp studies have shown that anti-porin antibodies, applied to the external side of excised plasma membrane patches of mammalian astrocytes, close chloride channels that are thought to be engaged in cell volume regulation. Frog oocytes are often used to study this basic cell function. Here we document the localisation of endogenous porin voltage-dependent anion-selective channels in Xenopus laevis oocyte plasma membranes. In confocal laser microscopy images a disjunctive pattern of fluorescing spots appear about 10 microm apart. Labelling was prevented by preabsorption of the antibodies with synthetic peptides comprising the epitope of the antigen. Immuno-gold marking of oocyte surfaces followed by silver enhancement of the gold particles lead to a plasma membrane labelling corresponding to that obtained by the confocal laser approach. The data suggests the presence of voltage-dependent, anion-selective channels in oocyte plasma membranes. This data should be borne in mind when frog oocytes are used to study the characteristics of endogenous or heterologously expressed ion channels or regulatory proteins.

Studies on human porin: XIII. The type-1 VDAC 'porin 31HL' biotinylated at the plasmalemma of trypan blue excluding human B lymphocytes.

In 1989, we demonstrated for the first time the expression of the VDAC 'Porin 31HL' in the plasmalemma of human B lymphocytes, then giving first evidence of a multi-topological expression of VDAC. Meanwhile, data from this and other laboratories support our proposal of a multi-compartment distribution of the channel in mammalian tissues. Here, by biotinylation of plasmalemma-integrated proteins of proven living B lymphocytes, followed by two-dimensional electrophoresis, immuno- and streptavidin affinity blotting, we show that part of the channel molecules can be labelled at the outer membrane of the cells. Thus, by a relevant approach our results invalidate objections concerning putative cross-reactivity of anti-human Type-1 porin antibodies with non-VDAC proteins at the outer cell membrane.

Golgi retention of human protein NEFA is mediated by its N-terminal Leu/Ile-rich region.

The subcellular localization of the human Ca(2+)-binding EF-hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti-NEFA monoclonal antibody 1H8D12 and rabbit anti-ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA-green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi-resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA-GFP variant was detected by fluorescence microscopy and immunoblotting. Only the DeltaN mutant, lacking the N-terminal Leu/Ile-rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF-hand pair domain, the two EF-hand motifs separately, the leucine zipper and the leucine zipper plus the C-terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile-rich region is discussed as a novel Golgi retention motif.

[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.

[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (Myeloma protein Tro). IV. Isolation and characterization of the chymotryptic peptides of the H-chain (author's transl)]. / Zur Strukturregel der Antikörper. Die Primärstruktur eines monoklonalen IgA1-Immunoglobulins (Myelomprotein Tro), IV. Isolierung und Charakterisierung der chymotryptischen Peptide der H-Kette.

The preceeding papers dealth with the purification and characterization of IgA immunoglobulin Tro and its H- and L-chains, the separation and characterization of the cyanogen bromide fragments and the isolation of the tryptic splitting products of the H-chain. In this paper the chymotryptic peptides of the H-chain are presented. All chymotryptic peptides were isolated and purified parallel to the isolation of tryptic peptides. On the basis of these overlapping splitting products the arrangement of the tryptic peptides in the protein could be determined. These and other splitting products listed in this paper contributed to the complete sequence determination of of the H-chain.

[The primary structure of a crystalline monoclonal immunoglobulin kappa-type L-chain, subgroup I (Bence-Jones protein Rei.); isolation and characterization of the tryptic peptides; the complete amino acid sequence of the protein; a contribution to the elucidation of the three-dimensional structure of antibodies, in particular their combining site (author's transl)]. / Die Primärstruktur einer kristallinen monoklonalen Immunglobulin-L-Kette vom kappa-Typ, Subgruppe I(Bence-Jones Protein Rei.), Isoleirung und Charakteristierung der tryptischen Peptide; die vollständige Aminosäuresequenz des Proteins. Ein Beitrag zue Aufklärung der räumlichen Struktur der Antikörper, insbesondere der Haftstelle

Bence-Jones protein Rei. was isolated from the urine of a plasmacytoma patient by precipitation with ammonium sulfate and purified by ion exchange chromatography. The determination of the molecular weight showed that the protein was present in its monomeric form. The preparation cotained a minor fraction, which consisted of the dimer of the variable parts and was characterized by its ability to crystallize. X-ray data with a resolution on the atomic level could be achieved and allowed, together with the known primary structure, the construction of a three-dimensional model, which also gave an insight into the antigen binding site. It consists of the hypervariable regions, which form a pocket 15 A in diameter lying between the two monomers at the ends of the molecules. Sequence studies were performed with tryptic peptides which were purified by chromatographic procedures and aligned in homology to other kappa-chains. The protein belongs to subgroup I on the basis of its characteristic amino acid sequence and follows a highly regular pattern of amino acid exchanges. Its sequence is in agreement with an evolutionary origin of antibody variability.

[The primary structure of a monoclonal IgA-immunoglobulin (IgA Tro), I: The amino acid sequence of the L-chain of lambda-type, subgroup II (author's transl)]. / Die Primärstrukur eines monoklonalen IgA-Immunoglobulins (IgA Tro.), I. Die Aminosäuresequenz der L-Kette, lambda-Typ, Subgruppe II

The primary structure of a monoclonal human IgA immunoglobulin has been determined. The protein has been isolated from the serum by salt fractionation, ion exchange chromatography and gel filtration. The L-chain was isolated by gel filtration after partial reduction and alkylation of the IgA-molecule. The primary structure of the L-chain was determined by sequence studies of its tryptic peptides. The protein containes 216 amino acid residues. Based on its homology with other proteins of known structure, the variable part of the protein clearly belongs to subgroup II of the lambda-chains. The constant part of the chain is Kern- and Oz-, which means that it has serine in position 154 and arginine in position 191.

[Pattern of antibody structure. The amino acid sequence of a monoclonal immunoglobulin L-chain of lambde-type, subgroup I (Bence-Jones-protein Vor.). A contribution to the elucidation of the origin of antibody specificity (author's transl)]. / Zur Strukturregel der Antikörper. Die Aminosäuresequenz einer monoklonalen immunglobulin-L-Kette vom lambde-Typ, Subgruppe I (Bence-Jones-Protein Vor.) Ein Beitrag zur Aufklärung der Entsthung der Antikörperspezifitäten

The experiments leading to the determination of the primary strucutre of Bence-Jones-Protein Vor. are reported. The variable part of this immunoglobulin polypeptide chain can easily be identified as a typical representative of subgroup I of the lambde-chains. The constant part is characterized by the Kern- and OZ+ markers. The sequence data of this protein are in complete agreement with the demands of the germ line theroy of antibody formation. To demonstrate the evolutionary origin of the V-henes, a phylohenetic tree of all completely sequenced Vk-, Vlambde-, and VH-chains is constructed. Teh evolutionary rate of immunoglobulins is calculated and compared to that of other protein families.

[The primary structure of a monoclonic immunoglobulin-L-chain of subgroup IV of the kappa type (Bence-Jones protein Len.)]. / Len.)Len

The complete amino acid sequence of Bence-Jones protein Len. was established by sequential analysis of tryptic and chymotryptic peptides. The result of these experiments and the comparative sequence analysis with the other 17 completely determined kappa-proteins is incompatible with the serological typisation of protein Len. as a member of subgroup II: There are 18 positions in protein Len. than cannot be associated with any one of the subgroups kappaI, kappaII or kappaIII. Also the average amino acid exchange rate between protein Len. and these subgroups is in the same range as the average amino acid exchange rate between these subgroups. Therefore Bence-Jones protein Len. is the first completely determined representative of a new IV. kappa-type subgroup. The variability of immunoglobulins follows a structural principle in which the single point mutations responsible for the variability are linked. The present paper contains the exact analysis of the linked point mutations within the so far best-investigated subgroup, kappaI (12 completely sequenced proteins). These linked exchanges allow the arrangement of the kappaI proteins in 4 subgroups and their further subdivision. The regularity of this amino acid sequence pattern can only be explained by an evolutionary origin of antibody variability. On the basis of this evolutionary mechanism the relationship of immunoglobulins can be depicted in a phylogenetic tree. Such a tree was therefore constructed for the 18 completely determined Bence-Jones proteins of kappa-type, for the first time taking into account Bence-Jones protein Len. Its topology is in complete agreement with the results of the comparative sequence analysis.

[Rule of antibody structure. The primary structure of a monoclonal IgG1 immunoglobulin (myeloma protein Nie), II: isolation and amino acid sequence of the tyrptic peptides from the H-chain (author's transl)]. / Zur Strukturregel der Antikörper. Die Primärstruktur eines monoklonalen IgG1-Immunoglobulins (Myelomprotein Nie), II. [1,2] Isolierung und Aminosäuresequenz der tryptischen Peptide der H-Kette

The reduced and carboxymethylated H-chain of protein Nie was digested with trypsin. 40 tryptic peptides were isolated from the digest by ion exchange chromatography, accounting for the entire amino acid composition of the polypeptide chain. Sequence studies performed with these fragments revealed the position of 444 out of 448 residues. In addition 8 overlapping cleavage products were purified which contributed to the ordering of tryptic peptides in the chain.

[The rule of antibody structure. The primary structure of a monoclonal IgG1 immunoglobulin (myeloma protein Nie). III. The chymotryptic peptides of the H-chain, alignment of the tryptic peptides and discussion of the complete structure]. / Zur Strukturregel der Antiköper. Die Primärstruktur eines monoklonalen IgG1-Immunoglobulins (Myelomprotein Nie), III. Die chymotryptischen Peptide der H-Kette, Anordnung der tryptischen Peptide und Diskussion der vollständigen Primärstruktur

In this final paper the complete primary structure of the H-chain of immunoglobulin Nie (IgG1, Gm1+, 17+) is established by overlapping tryptic fragments with chymotryptic peptides. The preceding papers dealt with the purification of the protein, the characterization of the light and heavy chains, the purification and characterization of the cyanogen bromide cleavage products, the location of the disulfide bonds, the isolation of the tryptic peptides and their sequence determination. The gamma1-chain Nie comprises 448 amino acid residues. When the protein is compared with other H-chains, the switch from the variable to the constant part occurs at position 119/120. Based on the amino acid sequence of the variable part, protein Nie belongs to subgroup III of the H-chains. It was the first protein of this subgroup to be sequenced. In the meantime several other proteins are known which have been assigned to the same subgroup on the basis of linked amino acid exchanges in comparison to members of other subgroups. This confirms the evolutionary origin of antibody variability and hence the genetically fixed antibody specific. Furthermore protein Nie is the first completely determined chain with the genetic factors Gm1+, 17+. These factors are inherited codominantly and are localized on the constant part of the gamma1-chain. By comparison with protein Eu, which is Gml-, 4+ and therefore an allele of Nie, these serologically defined factors are correlated with Eu. Besides the amino acid exchanges caused by the Gm-factors we elucidated a series of differences to the constant part of the protein Eu. These differences include 6 amide postions and the sequence from residues 387 to 391. Using the structure of IgG1 Nie as an example some rules for the evolution of immunoglobulin sequences have been described. In particular the "elongation-rule" and the "Disulfide-rule" are discussed. While chain-elongation of the H-chains can simply be explained by repeated gene duplications of a basic unit containing ca 110 amino acids, the location of disulfide bonds is determined partly by gene duplication, which implies multiplication of evolutionary "old" cystein residues and partly by the relatively recent acquisition of "new" cystein in appropriate sites. Most evident is the origin of the "hindge-region" by partial gene duplication on the C-terminal residues of the first homology region.

[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), III. Isolation and characterization of the tryptic peptides of the H-chain (author's transl)]. / Zur Strukturregel der Antikörper. Die Primärstruktur eines monoklonalen IgA1-Immunglobulins (Myelomprotein Tro), III. Isolierung und Charakterisierung der tryptischen Peptide der H-Kette.

The reduced and either aminothylated or carboxymethylated H-chain of the monoclonal IgA1 immunoglobulin Tro was digested with trypsin. The tryptic peptides were isolated by gel and ion-exchange chromatography. Because of different methods of alkylation, the cysteine-containing peptides could be obtained in two forms and showed additional overlaps. Sequence studies performed with these fragments elucidated the primary structure of the protein.

[Rule of antibody structure. Primary structure of a human monoclonal IgAl-immunoglobulin (myeloma protein Tro). VI. Amino acid sequence of the L-chain, lambda-type, subgroup II]. / Zur Strukturregel der Antikörper. Die Primärstruktur eines monoklonalen IgA1-immunglobulins (Myelomprotein Tro). VI. Die Aminosäuresequenz der L-Kette, lambda-Typ, Subgruppe II.

The primary structure of the L-chain of an IgA1-immunoglobulin (Myeloma protein Tro) has been determined by means of cleavage with trypsin and, if necessary, with alpha-chymotrypsin. The tryptic peptides of the variable part were characterized by amino acid analysis, Dansyl-Edman degradation and cleavage with carboxypeptidase; the peptides of the constant part were identified by amino acid analyses and determination of its N- and C-terminal residues. The sequence of the remaining amino acids and the arrangement of the peptides were established in homology to known structures. The protein comprises 216 amino acids. The homology of the variable part clearly characterizes it as belonging to subgroup II of lambda-chains. In positions 27a, b and c, there are the subgroup-specific additional residues and in position 96 is the characteristic deletion. The constant part of the chain is Kern- and Oz- which indicates that it has serine in position 154 and arginine in position 191.