Viswanathan Shankar

Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin

A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.

Polymeric structure of human respiratory mucin : studies on two protein components released upon reduction of disulfide bonds

A major mucus glycoprotein (mucin) was purified from the tracheobronchial secretions of an asthmatic patient. Upon SDS-composite gel electrophoresis, the purified native (non-reduced) mucin gave a single band. SDS-gel electrophoresis on 6% polyacrylamide gels showed the absence of low molecular mass protein contaminants. However, SDS-PAGE (6% gels) of the reduced mucin showed the presence of a major high molecular mass mucin component and two low molecular mass components of 118 and 70 kDa, respectively. The 118 and 70 kDa components were purified by preparative electroelution of the reduced mucin. These components were also separated from the reduced mucin by gel-permeation chromatography on a Superose 6 column. Chemical compositional analyses showed that the 118 kDa component was a glycoprotein while the 70 kDa component was non-glycosylated. The effect of disulfide bond reduction on mucin structure and the hydrophobic probe binding properties of native and reduced mucin were studied using the fluorescent probe technique. Mansylphenylalanine was used as the fluorescent probe. The native mucin showed the presence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the probe. On the other hand, reduced-alkylated mucin containing the 118 and 70 kDa components showed the presence of additional high-affinity (KD approximately 10(-6) M) binding sites as well as low-affinity binding sites for the probe. Reduced alkylated mucin devoid of the 118 and 70 kDa components showed the presence of only low-affinity binding sites. These observations suggest that the availability of high-affinity probe binding sites upon reduction of mucin disulfide bonds may be either due to binding of the probe to the released component(s) and/or due to noncovalent interaction of the released component(s) with the mucin causing a conformational change in the mucin structure. Thus, the 118 and 70 kDa components appear to be an integral part of the total polymeric structure of the human respiratory mucin.

Characterization of the Enterococcus faecalis Alpha C Protein Homolog

Enterococci have emerged as leading nosocomial pathogens3. Clinical isolates of enterococci are usually resistant to multiple antibiotics. Despite the threat posed by highly resistant strains of E. faecalis, little is known about the properties of this organism that mediate pathogenesis. Gram-positive bacteria express on their cell surface a variety of proteins that are involved in pathogenesis. Randomly obtained nucleotide sequences from the genome of an outbreak strain (MMH594)2 were screened for the hallmarks of cell surface proteins. One sequence was identified that exhibited sequence similarities with Streptococcus agalactiae Cα protein antigen1.

Biophysical characterization of mucin components HTM-1 and HTM-2 from tracheobronchial secretions of cystic fibrosis patients

Mucins present in the tracheobronchial secretions are responsible for the viscoelastic properties of the mucus. Any changes in the mucin structure may alter the physical properties of mucus and hence its function. Previous studies from this laboratory have reported the isolation and characterization of a major mucin component (HTM-1) and a minor, novel mucin component (HTM-2) from the tracheobronchial secretions of cystic fibrosis (CF) individuals. In the present study, the macromolecular properties of the CF mucin components HTM-1 and HTM-2 were further investigated using biophysical methods. Dynamic light scattering studies showed that CF HTM-1 and HTM-2 had a greater extended structure in buffer containing 0.10 and 0.15 M NaCl than that observed in the presence of 0.03 M NaCl. Also, CF HTM-1 had a compact configuration in the presence of 5 and 10 mM Ca2+, while under similar experimental conditions, the structure of CF HTM-2 was unaffected, indicating differences in the macromolecular properties of CF mucin components. Fluorescent probe binding studies revealed that CF HTM-1 had more hydrophobic probe binding domains than those observed for CF HTM-2. In summary, both biochemical and biophysical characterization suggests structural differences between the CF HTM-1 and HTM-2 components.

Nucleotide sequence of a cDNA for canine β-spectrin reveals high evolutionary conservation

A cDNA coding for the carboxy-terminus of canine beta-spectrin was isolated from a canine tracheal cDNA library. Analysis of the 3267 nucleotide sequence revealed a single open reading frame coding for 707 amino acids. Comparison of the deduced amino acid sequence to that of the recently reported general isoform of human beta-spectrin (beta G) revealed 98% identity. This high degree of conservation of the general isoform of beta-spectrin illustrates a strong evolutionary selection and should help in identification of sites that are candidates to mediate specialized functions of this general isoform. This is the first report of a cDNA encoding canine beta-spectrin.