Agnes Henschen

Tetanus toxin: primary structure, expression in E. coli, and homology with botulinum toxins.

A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75‐kbp plasmid from a toxigenic non‐sporulating strain of Clostridium tetani. The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol. wt of 150,700. In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol. wt light chain and the 98,300 mol. wt heavy chain, respectively. Cysteine (467) was involved in the disulfide linkage between the two subchains. The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C. tetani and C. botulinum are derived from a common ancestral gene. Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies. The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E. coli.

COVALENT STRUCTURE OF FIBRINOGEN

It is well established that the fibrinogen molecule is made up of two identical halves, each containing three different peptide chains. The overall structure may therefore be described as (Aa, Bp, y ) 2. On thrombin-digestion the fibrinopeptides A and B are released and fibrin, with the structure (a, p, yl2, is formed. The complete primary structure of human fibrinogen is known.l-16 The sequence information may be used to study relationships between structure, function and evolution. In genetically determined abnormal fibrinogens the correlation between the structural error and the dysfunction of the molecule may reveal the functional importance of single amino acid residues.

Opioid activities of β-casomorphins

Abstract β-Casomorphin-7 (H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH) and its analogues: β-casomorphin-6, (-5) and (-4) (derived by sequential removal of respectively one, two or three amino acid residues from the C-terminus), were tested for their opioid activities in a variety of assay systems. Each of the four peptides displayed opioid activity in an opiate receptor binding assay, the isolated mouse vas deferens (MVD), the guinea-pig ileum longitudinal muscle myenteric plexus preparation (GPI) and produced naloxone-reversible analgesia after intracerebroventricular injection into rats. In contrast, none of the peptides displayed opioid activity in the isolated rat vas deferens preparation (RVD). β-Casomorphin-5 was the most potent compound in all the assays employed. Each β-casomorphin was more potent on the GPI than on the MVD. In view of the fact that the GPI, MVD and RVD are populated predominantly by μ-, δ- and e-receptors, respectively, the β-casomorphins probably represent μ-type opiate receptor agonists.

Intracellular Accumulation of Insoluble, Newly Synthesized Aβn-42 in Amyloid Precursor Protein-transfected Cells That Have Been Treated with Aβ1–42

Our early study indicates that intracellular Aβ1–42 aggregates are resistant to degradation and accumulate as an insoluble residue in lysosomes, where they alter the normal catabolism of amyloid precursor protein (APP) to cause the accumulation of insoluble APP and amyloidogenic fragments. In this study, we examined whether the addition of exogenous Aβ1–42 also leads to the accumulation of newly synthesized intracellular Aβ. Here we describe that newly synthesized Aβ, especially Aβn-42, is generated from metabolically labeled APP and accumulates in the insoluble fraction of cell lysates after Aβ1–42 treatment. These results suggest that intracellular Aβ may derive from a solid phase, intracellular pathway. In contrast to the pathway that primarily produces secreted Aβ1–40, the solid-phase intracellular pathway preferentially produces Aβn-42 with ragged amino termini. Biochemical studies and amino acid sequencing analyses indicate that these intracellular Aβ also share the same types of Aβ structures that accumulate in the brain of Alzheimer’s disease patients, suggesting that a significant fraction of the amyloid deposits in Alzheimer’s disease may arise by this solid-phase pathway.

Acrosin shows zona and fucose binding, novel properties for a serine proteinase

The major fucose‐binding protein of 53 kDa from boar spermatozoa was isolated to apparent homogeneity using a two‐step procedure including high‐performance gel filtration and reversed‐phase chromatography. The N‐terminal sequence of the protein revealed that it is identical with the sperm proteinase acrosin. By means of a solid‐phase zona‐binding assay based on the avidin‐biotin system it was demonstrated that acrosin also interacts strongly with porcine zona pellucida. Thus, the acrosin molecule combines specific proteolytic activity with zona‐ and carbohydrate‐affinity properties, i.e. previously unrecognized properties of a serine proteinase. It seems likely that this special affinity of acrosin directs the proteolytic activity to its structural target in the in vivo situation.

Antinociceptive potencies of β-casomorphin analogs as compared to their affinities towards μ and δ opiate receptor sites in brain and periphery

Abstract β-Casomorphins and their analogs were tested for their opioid activities in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum (GPI), the isolated mouse vas deferens (MVD), and for their affinities to μ- δ- and κ-binding sites in rat brain membranes. C-terminal amidation of β-casomorphin-4 and (-5) increased opioid potency in both organ preparations (GPI, MVD) and affinity to μ-binding sites in brain whereas binding to δ-sites was diminished. These β-casomorphin-amides displayed a 2–3 times greater naloxone reversible antinociceptive effect than natural β-casomorphins. Introduction of D-alanine at position 2 in the β-casomorphin-amides increased potency in the GPI whereas activity in the MVD was only slightly changed. These compounds, however, showed a remarkable increase in binding to δ-sites in brain with an unaffected or slightly increased binding to μ-sites and decreased binding to κ-sites. D-Ala2-β-casomorphin-4 and (-5) amides were 10 times more potent antinociceptive agents than corresponding β-casomorphin-amides. These results suggest firstly, that peripheral δ-receptors in the MVD are not as closely related to δ-binding sites at rat brain membranes as is the case with μ-receptors in the GPI and μ-binding sites, and secondly, in addition to μ-receptors, δ-receptors may be of importance in mediating antinociception.

Large scale preparation of S-carboxymethylated chains of human fibrin and fibrinogen and the occurrence of γ-chain variants

Abstract The investigation aimed at preparing peptide chains of human fibrin and fibrinogen suitable, in quantity and chemical form, for determination of the primary structure. 10 g of fibrin or fibrinogen were mercaptolysed with mercaptoethanol, and subsequently carboxymethylated with iodoacetate, all in 9 M urea. The conditions used led to complete and selective conversion of cystine residues to S-carboxymethylcysteine residues. 5–10 g of S-carboxymethylfibrin were fractionated by chromatography on CM-cellulose in 8 M urea with a pH gradient 4.80 to 6.00. Three major and two minor components were isolated. N-terminal sequence determination allowed the identification of the major components as the α-, β- and γ-chain, and the calculation of the molecular weights 75 000, 68 000 and 52 000, respectively. Amino acid composition and electrophoretic mobility are given for each chain. The minor components, by their N-terminal sequence, were clearly related to the γ-chain, but differed in amino acid composition, and were therefore designated γx and γy. They constituted 1% and 6%, respectively, of total γ-chain. The fibrinogen derivative was fractionated in a similar way, and also produced five components. The three major components were identified as the α(A)-, β(B)- and γ-chain by sequence determination and thrombin digestion. Unexpectedly, thrombin released from the α(A)-chain not only fibrinopeptide A, but also a tripeptide, Gly-Pro-Arg. This could be placed in the sequence as following directly on the fibrinopeptide moiety. The minor components were tentatively identified with the γx- and γy-chains found in fibrin.

Purification and primary structure of murine cryptdin-1, a Paneth cell defensin

We have purified and determined the amino acid sequence of cryptdin‐1, a murine Paneth cell defensin. The peptide corresponds to a previously characterized mRNA that accumulates to high abundance during postnatal ontogeny of the small bowel. Acid‐extracted intestinal protein was fractionated by cation‐exchange chromatography and fractions were assayed for antimicrobial activity. One peak of anti‐Salmonella activity contained a putative defensin, based on its predicted electrophoretic migration in acid‐urea PAGE. The peptide was purified to homogeneity by RP‐HPLC and sequenced. These studies demonstrate defensin expression in non‐myeloid tissue. The N‐terminal extension of cryptdin‐1 is a unique structural feature of this novel epithelial defensin.

Structural relatedness ofκ-casein and fibrinogenγ-chain

Summaryκ-Caseins, involved in the milk clotting process, and human fibrinogenγ-chain, involved in the blood clotting process, show structural similarities. Several longκ-casein sections, together corresponding to 80% of the whole protein molecule, have their counterparts in theγ-chain of fibrinogen, in that 31-42% of the amino acid residues occupy identical positions. The section ofК-casein which contains the chymosin-sensitive bond has a counterpart not only in theγ but also in the Bβ-chain of fibrinogen. Furthermore, the secondary structures of theК-caseins and of theγ-chain predicted according to the method of Chou and Fasman present several common features.

Identification and characterization of the genes encoding three structural proteins of the Sulfolobus virus-like particle SSV1

SummaryThree structural proteins, VP1, VP2 and VP3, of the virus-like particle SSV1 of the thermoacidophilic archaebacterium Sulfolobus sp. strain B12 were purified. VP1 and VP3 are very hydrophobic and show a high degree of homology. They consist of 73 and 92 amino acid residues, respectively. The third protein, VP2, is extremely basic containing 29 basic amino acids but only 4 acidic ones in a total of 74 amino acid residues. The genes encoding these three proteins were mapped within the genome by comparison of N-terminal amino acid sequences with the SSV1 DNA sequence. The three genes are closely linked in the order VP1-VP3-VP2 and the coding strand is the same in all three genes. Ten nucleotides separate the stop codon for VP1 from the initiation codon for VP3 and one nucleotide separates the genes encoding VP3 and VP2. Duplicate putative ribosome binding sites are found upstream of the initiation codons for VP2 and VP3. The major coat protein VP1 does not start with a methionine residue and appears to be the result of proteolytic cleavage of a precursor molecule. Transcription of the region encoding VP1, VP2 and VP3 results in the formation of two mRNAs of 0.5 kb and 1.0 kb, the shorter one only encoding VP1, the longer one spanning all three genes. A 61 bp sequence encoding part of VP1 is exactly repeated within the gene for VP3 and these identical sequences are translated into stretches of identical amino acids in the two proteins. A function of this repeated DNA sequence beyond its coding properties is very likely.