Biomedical subjects

N Hilschmann

Publications and source records attributed to N Hilschmann.

At least 91 records · Page 5Linked to original sources

PubMed1976

[Rule of antibody structure. the primary structure of a monoclonal IgG1 immunoglobulin (myeloma protein Nie), I: Purification and characterization of the protein, the L- and H-chains, the cyanogenbromide cleavage products, and the disulfide bridges (author's transl)].

Myeloma protein Nie has been isolated from the serum of a myeloma patient by free flow continuous high voltage electrophoresis or by Pevicon-block electrophoresis. It was further purified by ion-exchange chromatography and gel filtration, and characterized by amino acid analysis and end group determination. Serologically, the protein belongs to the IgG1 subclass. It has been typed as Gm1+, 2-,4- and 17+. The L-chain is of the k-type. The L- and H-chains have been separated by gel-filtration after partial reduction and alkylation and characterized by amino acid analysis and end group determination. The F(ab)- and Fc-fragments, prepared by limited tryptic digestion, have been separated and characterized. Cyanogen bromide splitting products have been prepared both from the intact IgG and from the Fc-and the partially reduced and alkylated F(ab)-fragment. These splitting products have been purified and characterized by amino acid analysis and end group determination. By means of these cyanogen bromide splitting products and by partial reduction and alkylation, the disulfide bridges in the protein could be localized: one L-H-bridge, two inter-H-bridges and four loop forming intra-H-bridges.

Alkylation↗

PubMed1976

[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)].

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.

Amino Acid Sequence↗

PubMed1976

[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].

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.

Amino Acid Sequence↗

PubMed1975

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

Amino Acid Sequence↗

PubMed1975

[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)].

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.

Amino Acid Sequence↗

PubMed1975

[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)].

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.

Amino Acid Sequence↗

PubMed1975

[The primary structure of a monoclonal IgA-immunoglobulin (IgA Tro.), II. The amino acid sequence of the H-chain, alpha-type, subgroup III; structure of the complete IgA-molecule (author's transl)].

The primary structure of a monoclonal human IgA-immunoglobulin has been determined. Sequence studies were carried out with the isolated L-chain [1], the isolated H-chain and with BrCN-fragments of the H-chain. Tryptic and chymotryptic peptides were prepared from the totally reduced and alkylated alpha-chain or from BrCN-fragments. Sequence work has been done with tryptic as well as chymotryptic peptides. The variable part comprised positions 1-119, the constant part residues 120-472. According to its homology with other variable parts alpha-chain Tro. clearly belongs to subgroup III of the H-chains. The constant part is composed of 3 homology regions (C1-C3) which originated from a common ancestor by repeated gene duplications early in evolution. Each homology region corresponds in its length and its sequence to the C-region of the L-chains. The hinge-region, which connects the C1- and the C2-region, originated from the C-terminal end of the C1-region by a twofold partial gene duplication comprising 8 amino acids. The C3-homology region is termined by an additional C-terminal piece of 18 residues. The alpha-chain 17 cysteine residues, 8 of which form the usual intrapeptidal loop S-S-bridges, and one the connection between the L- and H-chain. Two additional cysteine residues are located in the C1-region, and 5 more in the C2-region, forming intra- and inter-peptidal S-S-bonds.

Amino Acid Sequence↗

PubMed1975

[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)].

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.

Amino Acid Sequence↗

PubMed1974