MacDNAsis Analysis of
Lysozyme Genbank search results


Using the lysozyme cDNA and amino acid sequences from five different organisms (fly, mouse, E. coli, chicken, and human) obtained in a previous Genbank search of lysozyme, the following page is a presentation of a MacDNAsis analysis. MacDNAsis is a computer application which analyses either DNA or amino acid sequences and produces a myriad of useful information. In this analysis, the predicted open reading frame of the fly cDNA sequence was first determined. Then using this ORF, the molecular weight of lysozyme was calculated, hydropathy and antigenicity plots were produced, a visual representation of the secondary structure was made, and finally the similarity of the five amino acid sequences was determined through a multiple sequence analysis and a proposed phylogenetic tree. The results are summarized below.

1. Open Reading Frame (ORF) Determination

Using the 1366 nucleotide fly (Drosophila melanogaster) lysozyme cDNA sequence obtained through the previous Genbank search, the largest open reading frame was determined. First the plot below (Fig. 1)was produced. This plot revealed the largest open reading frame (shaded in black) which is assumed to be the coding region for fly lysozyme.
Fig. 1. MacDNAsis analysis of open reading frame (ORF) for fly (Drosophila melanogaster) lysozyme. The numbers along the top indicate the nucleotide number. The three different rows represent the three different reading frame possibilities of a codon sequence. Start codons (ATG) are represented by the orange triangles, stop codons (TAA, TAG, TGA) are represented by the green vertical bars, and the white areas represent ORFs. The black box in the top row is simply a highlight of the largest ORF. We assumed that this largest ORF was the coding region of the lysozyme protein.

2. Molecular Weight (MW) Determination

The sequence location of this ORF was then determined (aa's 634-1119). After translation, further analysis revealed that this region encodes a 18.066 kDa protein (data not shown).

3. Determination of Lysozyme Hydropathy

The fly ORF was then analyzed using a Kyte and Doolittle plot (Fig. 2). This plot shows both hydrophobic and hydrophilic regions of the fly lysozyme protein. Since protein membrane spanning domains are predominantly hydrophobic, the Kyte and Doolittle plot is used to predict such regions.

Fig. 2. A Kyte and Doolittle hydropathy plot of fly (Drosophila melanogaster) lysozyme ORF. The x-axis shows the amino acid number in the sequence; positive y-axis values are hydrophobic and negative values are hydrophilic. The region centered near aa 30 displays significant hydrophobicity.

The significantly hydrophobic region near aa 30 indicates that this region could possibly span a membrane. However, since the remainder of the molecule is significantly hydrophilic, it seems unlikely that lysozyme is an integral membrane protein. Also given that lysozyme is believed to be cytoplasmic, it seems then, that this hydrophobic domain is rather an indication of a hydrophic folding region (as will be discussed in section 5).

4. Determination of Lysozyme Antigenicity

Next, a Hopp and Woods plot (Fig. 3) was generated to determine hydrophobic--and therefore potential antigenic--regions in the lysozyme protein. Antibody-antigen interactions are dependant upon electrostatic forces, hydrogen bonding, van der Waals interactions, and hydrophobicity1. It is these specific interactions which give antibodies their high specificity. Thus when wanting to raise monoclonal antibodies against a protein, for the purpose of producing a probe, it is necessary to determine what region will provide the most successful epitope.

Fig. 3. Hopp and Woods antigenicity plot of fly (Drosophila melanogaster) lysozyme ORF. X-axis shows amino acid numbers; positive y-axis values are hydrophobic and negative values are hydrophilic. The region surrounding aa 50 displays a relatively hydrophobic region.

Fly lysozyme appears to have two relatively weak hydrophilic regions and two relatively weak hydrophobic regions. The largest of these hydrophobic regions is the one surrounding aa 50. Due to the hydrophobicity, this region will display good antigenicity. Therefore, one would want to sequence this region and raise antibodies against it when producing a probe against lysozyme.

5. Determination of Predicted Secondary Structure

Next, the secondary structure of the fly lysozyme ORF was predicted using MacDNAsis. There are four levels of protein structure: primary, secondary, tertiary, and quaternary. Primary structure refers to the actual amino acid sequence that comprise a protein. Secondary structure refers to the local spatial arrangement of the amino acids, resulting in alpha-helices or beta strands. Tertiary structure is the 3-dimensional folding of the subunit due to forces such as disulfide bonds. Finally quaternary structure refers to interaction of separate subunits to produce a whole protein. MacDNAsis analysis produced the following diagram of predicted secondary structure for fly lysozyme (Fig. 4).

Fig. 4. Chou, Fasman, and Rose analysis predicting the secondary structure for fly lysozyme (aa 1-162). As indicated in the legend, blue bars represent alpha-helices, red striped bars represent beta-strands, green bars represent turns in the structure, and black-checkered bars represent coiled domains.

From this prediction, fly lysozyme contains four alpha helices, eight beta pleated sheets, six coiled domains, and two major turns.

When compared to a RasMol image of lysozyme, this predicted structure seems relatively consistent. The RasMol image (viewed best in Display:ribbons) appears to have four alpha-helices and three beta-strands (forming a beta-pleated sheet) separated by coiled domains. In addition, when tracing the sequence, the RasMol structure does seem to illustrate two large turns just after the first two alpha helices and the three beta-strands. Thus with the exception of the unseen beta strands at the terminal half of the molecule, the MacDNAsis generated secondary structure image and the RasMol image seem to confirm the structure of lysozyme.

6. Multiple Sequence Alignment

The five amino acid sequences obtained through the previous Genbank search were analysed for sequence similarity. Recently, sequence analysis has provided another powerful tool for determining evolutionary relationships. Fig. 5 show each lysozyme ORF from the five organism (fly, E. coli, chicken, human, and mouse).

Fig 5. Lysozyme amino acid sequence alignment for five organisms (Dmelanogaste-fly, EcoligsnAA-E. coli, GgallusgsnAA-chicken, HsapiensgsnA-human, and Mmusculusgsn-mouse). Number to the left and right of the sequences and above each sequence group indicate amino acid number. Letters represent amino acids, with dashes (-) inserted to maximize sequence alignment. Black boxes indicate amino acid conservation.

From this figure, it appears that human lysozyme and mouse lysozyme are most closely related. This is seen particularly in the fourth block of sequences (151-200), as the greater portion of the aa's are conserved between the two.
On a more puzzling note, none of the first 35 aa's in the human lysozyme are conserved. Although it is difficult to determine the most primitive sequence, the human lysozyme sequence seems to have mutated significantly from it. Whether there is a function significance or an evolutionary significance to the human protein, is equally difficult to tell, but a sequence comparison on a much larger scale might provide more insight.

7. Proposed Phylogenetic Tree

From the sequence alignment produced in Fig. 5, a phylogenetic relationship for lysozyme was constructed.

Fig. 6. Phylogenetic tree showing the determined sequence conservation among the five organisms (HsapiensgsnA-human, MmusculusgsnA-mouse, Dmelanogaste-fly, EcoligsnAA-E. coli, GgallusgsnAA-chicken). Numbers indicate lysozyme percentage aa sequence conservation between shown organisms.

This proposed tree confirms the close relation of the human, mouse, and to a lesser degree, fly, E. coli, and chicken lysozyme proteins. A large homology is seen between human and mouse lysozyme with 74.6% of the residues conserved. Next closest in relationship is the fly lysozyme with 24.3% conserved between it and the other two, followed by the E. coli lysozyme, having a 7.5% conservation rate. Finally, the chicken lysozyme showed the least rate of conservation, only 6.7%.
It is clear that this tree is not wholly consistent with evolutionary lineage. One would expect that the mouse would be most conserved, followed by the chicken, the fly, and E. coli. A plausible explanation for this is the selection pressures exerted on domesticated chickens. Since domesticated chicken have a high exposure rate to bacterial infection, it is consistant that a defense mechanism, such as lysozyme, might be highly mutated. Thus in species where there is less selection pressure, one expects the lysozyme protein to be more conserved. This is largely seen in the phylogenetic tree. Nonetheless, this aberration demonstrates the precarious nature of sequence analysis in predicting phylogenetic relationship.


1. Stryer L. 1995. Biochemistry. 4th ed. New York: W.H. Freeman and Company. p 372.

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