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Clostridium botulinum types A, B, C1, and E produce proteins with or without hemagglutinating activity: do they share
1Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706.
Abstract:
Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C1, and E) and simultaneously proteins, A Hn+, B Hn+, C Hn+, and E Hn-, that have high, low, and no hemagglutinating activity. A Hn+ and B Hn+ are serologically cross-reactive. A Hn+, B Hn+, and C Hn+ found as large aggregates (900-220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn+, B Hn+ is 17 kD and 27 kD for C Hn+. The 116 kD E Hn- does not aggregate. We determined the sequences of 10-33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn+ and B Hn+. Each of these subunits have unique sequences, indicating that the larger units studies are not homomers or heteromers of smaller units. The subunits of A Hn+ and B Hn+ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity). In vitro proteolysis of 116 kD E Hn- with different proteases did not impart hemagglutinating activity to the fragments. The 116 kD E Hn- and one of its proteolytic fragments (87 kD) were partially sequenced. Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn+, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzuki et al., 1990). The derived amino acid sequence of this open reading frame, we found, has 73-84% sequence identity with those of the 17 kD subunits of A Hn+ and B Hn+ and significant identity with the N-terminal of E Hn-. These highly conserved sequences show existence of genetic linkage among the Hn+ and Hn- proteins.
Insights
Clostridium botulinum produces distinct neurotoxins and hemagglutinin proteins. Analysis reveals conserved genetic sequences among these proteins, suggesting a linkage between hemagglutinin-positive and hemagglutinin-negative variants.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Chemistry
Background:
- Clostridium botulinum synthesizes antigenically distinct neurotoxins and associated hemagglutinin proteins (Hn+ and Hn-).
- Hemagglutinin activity varies (high, low, none) among these proteins.
- Certain hemagglutinin proteins (A Hn+, B Hn+) exhibit serological cross-reactivity.
Purpose of the Study:
- To investigate the subunit composition and sequence homology of Clostridium botulinum hemagglutinin proteins.
- To explore the genetic relationship between hemagglutinin-positive and hemagglutinin-negative proteins.
Main Methods:
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to dissociate protein aggregates.
- Amino-terminal residue sequencing of protein subunits.
- Partial sequencing of hemagglutinin-negative proteins and their proteolytic fragments.
- Analysis of open reading frames downstream of cloned genes.
Main Results:
- Large aggregates of A Hn+, B Hn+, and C Hn+ dissociate into multiple subunits, with distinct sequences for each subunit size.
- Subunits of A Hn+ and B Hn+ of comparable molecular weight show high sequence identity.
- Proteolysis of E Hn- did not induce hemagglutinating activity.
- An open reading frame downstream of C Hn+ shows significant sequence identity to subunits of A Hn+, B Hn+, and E Hn-.
Conclusions:
- The larger hemagglutinin protein units are not simple homomers or heteromers of smaller observed subunits.
- Highly conserved sequences suggest genetic linkage between hemagglutinin-positive and hemagglutinin-negative proteins of Clostridium botulinum.