Biomedical subjects

N Hilschmann

Publications and source records attributed to N Hilschmann.

At least 73 records · Page 4Linked to original sources

PubMed1983

Primary structure of human class II histocompatibility antigens 3rd communication. Amino acid sequence comparison between DR and DC subclass antigens derived from a lymphoblastoid B cell line homozygous at the HLA loci (HLA-A3,3; B7,7; Dw2,2; DR2,2: MT1,1; Dc1,1: MB1,1).

Class II histocompatibility antigens from a homozygous human lymphoblastoid B cell line (HLA-A3,3; B7,7; Dw2,2; DR2,2; MT1,1; DC1,1; MB1,1) were isolated by an exclusively chemical procedure. After the separation of the alpha and beta chains the amino acid sequences of the extracellular parts of the DR2 alpha and the DR2 beta chains could be determined, in spite of the heterogeneity of both fractions. Peptides isolated in considerably lower yields indicated at least one more alpha chain and 6 further beta chains. By the investigation of these lower-yield enzymatic fragments, in addition to the DR2 alpha and DR2 beta chains, we can now report on the partial amino acid sequences of a DC alpha chain, a second beta chain of DR type and one beta chain of DC type. Furthermore, the isolation of a characteristic glycopeptide suggests the existence of a second beta chain of DC type. The following class II antigens are therefore identified: DR2 alpha/DR2 beta 1, DR2 alpha/DR2 beta 2, DC1 alpha/DC1 beta 1, DC1 alpha/DC1 beta 2, indicating that each subclass can be subdivided into two antigens because each alpha chain type can associate with at least either beta 1 or beta 2. Further investigations must clarify whether the remaining three beta chains are of the DR, DC, or of a third beta chain type. Amino acid sequence comparisons confirm that the human DR antigens are the equivalent of mouse I-E antigens, while human DC antigens are the equivalent of mouse I-A antigens.

Amino Acid Sequence↗

PubMed1983

[Is the HLA-DR associated glycoprotein p31 a ubiquitous molecule?].

The glycoprotein p31 (also called Ii, In, M1, Dr gamma, XM 1) has been shown up to now only in the membranes of B lymphocytes as well as of epidermal and endothelial cells, where it is always accompanied by antigens of the HLA-DR type. It is therefore called HLA-DR-associated protein. As we show here, the membranes of muscle, liver and brain contain protein molecules with the relative molecular mass Mr = 31 000-33 000 and an isoelectric point around 7.5. These parameters correspond to those of the p31 of B lymphocytes. These molecules, as well as the p31 of B lymphocytes, can be concentrated by ion exchange chromatography. In two-dimensional electropherograms they are identical to those of B lymphocytes. It can therefore be assumed that the po 31 is not really associated with the HLA-DR antigens but is a ubiquitous molecule.

B-Lymphocytes↗

PubMed1983

[Three-dimensional structure determination of antibodies. Primary structure of crystallized monoclonal immunoglobulin IgG1 KOL, I].

The crystallizable myeloma immunoglobulin IgG1 KOL [allotype Gm(-1,4), (gamma 1, gamma)2] which is well characterized in its three-dimensional structure by X-ray diffraction analysis of high resolution has been proved to be homogenous by polyacrylamide gel electrophoresis. The H- and L-chains were separated by gel filtration after complete reduction and carboxymethylation and were characterized by amino acid analysis, end group determination and polyacrylamide gel electrophoresis, respectively. The intact IgG1 KOL was cleaved by cyanogen bromide and all CNBr-fragments were isolated and characterized. The reduced and carboxymethylated H-chain was digested by trypsin and the tryptic hydrolysate was separated by ion-exchange chromatography. Using different procedures of rechromatography 35 out of 37 tryptic H-chain peptides could be isolated in sufficient amounts, the missing 2 peptides were produced by tryptic digestion of 2 CNBr-fragments. The amino acid sequences of all tryptic peptides were determined using a modified Edman degradation method after separation of the enzymatic cleavage products by high-performance liquid chromatography (HPLC). The complete primary structure of the VH-part of the H-chain was established by isolation and partial sequence determination of overlapping peptides obtained from cleavage of the intact H-chain by Staphylococcus aureus proteinase. The gamma 1-H-chain KOL comprises 455 amino acid residues and belongs to subgroup III. The switch from the variable to the constant part occurs at position 126/127, thus making VH-KOL one of the longest variable parts among the yet known immunoglobulin H-chains. This is due to the hypervariable region Hhv4 which is made up by 17 amino acid residues (4-9 residues more compared with other VH-parts). Within this region a so far not described additional intrapeptidal disulfide bridge could be localized (Cys 105-Cys 110) that creates a short loop with antiparallel running peptide strains in beta-pleated sheet conformation. Its role in the three-dimensional structure of the antigen-binding site of the IgG1 KOL molecule is discussed using the data obtained from X-ray diffraction analysis.

Amino Acid Sequence↗

PubMed1982

The role of gene translocation in the determination of specificity and class of the antibody molecule.

Structural studies of monoclonal immunoglobulins demonstrated that immunoglobulin chains are organized in the antigen-binding variable (V) part and the constant (C) part. The variability pattern indicated the separate genetic control of V and C parts. The decisive steps in B cell differentiation are gene translocation and fusion events which assemble V and C genes to the actual one active H chain gene and the one active L chain gene in a B cell or plasma cell. Further gene translocations, now solely on the H chain C genes, effect the H chain class switch. By DNA structure analysis of immunoglobulin genes from stem cells and plasma cells, the details of the internal organization of the genes and of the gene translocations were recognized. RNA processing of H chain precursor mRNA decides whether the immunoglobulin molecule is produced as an antigen receptor or an antibody molecule. It also enables the simultaneous expression of antigen receptors as IgM and IgD molecules on the B cell membrane.

Amino Acid Sequence↗

PubMed1982

[Primary structure of class II human histocompatibility antigens. 2nd Communication. Amino acid sequence of the N-terminal 179 residues of the alpha-chain of an HLA-Dw2/DR2 alloantigen (author's transl)].

From a lymphoblastoid homozygous cell-line (HLA-A3,3; B7,7; Dw2,2; DR2,2) the alpha-chain of the HLA-Dw2/DR2 antigen was isolated by an exclusively chemical procedure. The alpha- was separated from the beta-chain by chromatography with hydroxylapatite in Na-dodecyl sulfate. Here we describe the amino acid sequence of the alpha-chain up to Position 179. The molecule is divided into two domains which do not appear homologous to each other but show a significant homology to the beta-chain (21.2%). The similarity is larger in the second (27%) than in the first (15.5%) domain, indicating a different evolutionary relationship for both parts. In contrast to the beta-chain both domains contain an N-glycosidically linked carbohydrate. The methionines are positioned only in the N-terminal, the cysteines exclusively in the C-terminal domain. Only the latter can therefore be stabilized by a disulfide bridge. As with the beta-chain the regions around the cysteines show a remarkable similarity with the constant C-terminal domains of k-, lambda-, alpha-, gamma-, sigma-, epsilon- and mu-chains of immunoglobulins. Although to a considerably lesser extent, the alpha-chain preparation also shows a heterogeneity at the protein level. Since the employed cell-line is homozygous with regard to HLA-D/DR, our results indicate that at least two alpha-chain genes exist in the HLA-D/DR-region. Together with the already published sequence of the beta-chain, the extracellular part of an histocompatibility antigen of the HLA-D type is now known.

Amino Acid Sequence↗

PubMed1981

[Chromatography and rechromatography in high-performance liquid chromatography of peptide mixtures: the complete primary structure of an immunoglobulin L-chain of kappa-type, subgroup I (Bence-Jones Protein Den) (author's transl)].

In continuation of our work on the separation of enzymatic hydrolysates by high-pressure liquid chromatography with reverse-phases we present two new buffer systems: firstly 0.005M potassium phosphate, pH 6.0 and secondly aqueous trifluoroacetic acid, pH 2.15. As organic solvent acetonitrile is always used. The excellent properties are demonstrated by the separation of the tryptic peptides of Bence-Jones protein Den (Mr = 23000). Like ammoniumacetate these buffers are well suited for the first separation of peptide mixtures but can be used rather effectively in rechromatographies. If peaks are not or not fully resolved during the first chromatography they can be separated by a rechromatography in one of the other systems. This was most successful since in both cases the same high resolving technique is employed. From the primary separation 14 peptides could be isolated in analytically pure form. The remaining fragments were completely purified after one rechromatography. The amino acid analysis yielded also integer numbers and by a modified Edman degradation the primary structure could be determined. As with protein Wes (Kratzin, H., Yang, C.Y., Krusche, T.U. & Hilschmann, N. (1980) Hoppe-Seyler's Z. Physiol. Chem. 361, 1591--1598) all the peptides were recovered after high-pressure liquid chromatography, none was missing. From these data and from homology reasons the complete amino acid sequence of protein Den could be established. It contains 214 residues and belongs to subgroup I of the kappa-chains. The valine residue in position 191 indicates that it belongs to allotype Inv b+.

Amino Acid Sequence↗

PubMed1981

[Primary structure of class II human histocompatibility antigens. 1st communication. Amino acid sequence of the N-terminal 198 residues of the beta chain of a HLA-Dw2,2;DR2,2-alloantigen (author's transl)].

The beta-chains of HLA-D/DR antigens from a lymphoblastoid homozygous cell line (HLA-A3,3;B7,7;-Dw2,2;DR2,2) were purified by an exclusively chemical three step procedure: 1) Nonidet P-40/CM-cellulose ion-exchange-, 2) Na-dodecyl sulfate/sephacryl S-300- and 3) Na-dodecyl sulfate/hydroxylapatite chromatography. The amino acid sequence of the beta chain was established up to position 198. These residues comprise the extra-cellular part of the molecule which consists of two domains each probably containing a disulfide bridge, like immunoglobulin L chains. Within the first domain the carbohydrate moiety is attached to asparagine in position 19. Sequence work was hampered by the fact that the beta chain preparation of the HLA-Dw2,2;DR2,2 antigen actually consists of a pool of beta chains. The beta chain, the sequence of which has now been established, represents about 70% of the beta chain pool. The remaining 30% consist of at least 6 more chains which are homologous among one another as well as to the described beta chain. Since the antigens were gained from a homozygous lymphoblastoid cell-line our results indicate that the HLA-D region codes for at least 7 beta-chains and must therefore consist of a cluster of beta chain genes.

Amino Acid Sequence↗

PubMed1980

The primary structure of the constant part of mu-chain-disease protein BOT.

The complete primary structure of the constant part of the mu-chain-disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. two amino acid changes were found, at positions 309 (Ser leads to Gly) and 333 (Val leads to Gly) (GAL numbering). In two additional monoclonal mu chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of mu chains exist in the human: (1) GAL type with Ser-309 and Val-333; (2) OU type with Gly-309 and Val-333; (3) SCO type with Ser-309 and Gly-333; (4) BOT/CO type with Gly-309 and Gly-333. The meaning of this molecular polymorphism is discussed.

Amino Acid Sequence↗

PubMed1980

[Preparative separation of the tryptic hydrolysate of a protein by high-pressure liquid chromatography. The primary structure of a monoclonal L-chain of k-type, subgroup I (Bence-Jones Protein Wes) (author's transl)].

The tryptic hydrolysate of Bence-Jones protein Wes (Mr = 23000) was separated by high-pressure liquid chromatography in a volatile buffer system on a reversed-phase column. From the resulting 22 peptides 18 yielded integer numbers after amino acid analysis. They could be used directly for a modified Edman degradation. Two peptides were not separated by this procedure, two were missing. Under different conditions these four peptides could also be prepared in pure form. 3 mg was sufficient to elucidate the primary structure of the variable part of this protein completely. The arrangement of the tryptic peptides was deduced by homology to other k=chains. Protein Wes contains 214 residues and belongs to subgroup I of the k-chains. The valine residue in position 191 indicates that it belongs to allotype Inv b+.

Bence Jones Protein↗

PubMed1979

[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].

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.

Amino Acid Sequence↗

PubMed1979

[Rule of antibody structure. Primary structure of a human monoclonal IgA-immunoglobulin (myeloma protein Tro). VII. Purification and characterization of the disulfide bridges].

Myeloma Protein Tro has been isolated from the plasma of a myeloma patient. Monomeric IgA was separated from its polymer (by chromatography on Sephadex G-200). Both the forms were split with pepsin or cyanogen bromide and, if necessary, with thermolysin and subtilisin. The cystin-containing peptides were isolated from the hydrolysates by chromatography on Sephadex, ion-exchange columns, preparative paper chromatography, thin-layer chromatography, electrophoresis or by a combination of these methods. They were characterized by amino acid analyses and by determination of the N-terminal amino acids using the Dansyl-Edman procedure. Thus all the disulfide bridges of an IgA1 immunoglobulin could be established. The monomer has all together 48 cysteins, seven in each L- and seventeen in each H-chain; all these are covalently bonded by SS-bridges. Free SH-groups were not detected. The J-chain could only be identified serologically in the polymeric form of the protein. It is shown that the subunits of the polymers are covalently attached through either Cysl3, Cysl7 or both these residues of the H-chain.

Amino Acid Sequence↗

PubMed1979

[Purification and characterization of the free secretory piece of human colostrum (author's transl)].

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.

Amino Acid Sequence↗

PubMed1978

Genetic determination of antibody specificity. Gene translocation and fusion, the molecular basis for the differentiation of the antibody-producing cell.

The best system for the study of cell differentiation is a cell which in its differentiated state differs only by one product. This is the case in the immune system. The undifferentiated, but omnipotent stem cell differentiates into a committed B cell which produces only one type of specific antibody out of a million different, genetically fixed possibilities. Gene translocation and fusion is the basis of this differentiation process.

Alleles↗

PubMed1978

[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), I. Purification and characterization of the protein and its L- and H-chains (author's transl)].

Myeloma protein Tro was prepared from the serum of a myeloma patient by ammonium sulfate precipitation. It was purified by gel filtration and ion exchange chromatography or by Pevikon block electrophoresis. The purity of the preparation was tested by several electrophoretic or immunoelectrophoretic methods. L- and H-chains of the purified protein after reduction and alkylation were separated by gel chromatography. The protein and its L- and H-chains were characterized by amino acid analyses and end-group determinations.

Amino Acid Sequence↗

PubMed1978

[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), II. Cleavage of the monomer IgA-molecule and the reduced and alkylated H- and L-chains by cyanogen bromide (author's transl)].

The monomer of myeloma protein Tro as well as the reduced and alkylated H- and L-chains were cleaved by cyanogen bromide. All cyanogen-bromide fragments were isolated and characterized by amino acid analyses, end-group and molecular weight determinations. The 4 smaller fragments of the 5 H-chain fragments were split with trypsin. The peptides were isolated and their primary structure was determined.

Amino Acid Sequence↗

PubMed1978

[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)].

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.

Amino Acid Sequence↗

PubMed1978

[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)].

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.

Amino Acid Sequence↗

PubMed1978

[Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), V. The arrangement of the tryptic peptides and a discussion of the complete primary structure of the H-chain (author's transl)].

This communication deals with the sequence work done with tryptic and chymotryptic peptides and some cyanogen bromide splitting products. With these peptides, and if necessary with their splitting peptides, the whole primary structure of the alpha1-H-chain of myeloma protein Tro is established. The position of the amides is determined by electrophoresis and digestion with aminopeptidase M. The alpha1-chain Tro comprises 475 amino acid residues. Because of its specific exchanges and deletions the variable part of alpha1-chain Tro belongs to subgroup III of variable parts of H-chains. The switch from the variable to the constant part occurs at position 119/120 and is analogous to other chains which have been sequenced up to now. The large number of cysteine residues in the alpha-chain which may influence the tertiary structure, especially in the hinge and the subsequent CH2-region, is noteworthy. Furthermore, myeloma protein Tro is compared with the other alpha1-chain Bur[5] sequenced in the meantime, and protein But[6], which is an IgA2 molecule of the allotype A2m(2).

Amino Acid Sequence↗