Year: 1996
Type: dissertation
Abstract: This work extends research directed at the structural and functional characteristics of the lectin(s) from grasshopper Melanoplus differentialis and their role in immunity and molecular defense.Insect lectins mediate the progression of cellular defense and are considered a form of primordial recognition molecule.These proteins represent a potential immuno-recognitory surveillance molecule of broad carbohydrate specificity and their biochemical and genetic characterization are prerequisite to a complete understanding of invertebrate immune defense.Seventeen (17) clones have been isolated from a cDNA library constructed from fat body mRNA.Three clones were sequenced (Clones 2, 3 and 4).The sequences for clones 2 and 3 represent the same mRNA.Clone 3 was 879 bp long, but missing ≈ 200 bp at the 3' end including the stop codon and polyA tail.The clone 4 cDNA is 1079 bp long and includes sequence representing a stop codon and the 3' non-translated region through the polyA tail, but is incomplete at the 5' end missing sequence representing the initiating Met and signal sequence.Comparison of these sequences indicates the two are similar but represent distinct mRNAs, and presumably distinct genes.PCR evaluation of the remaining 14 clones, using gene-specific primers, has determined that a clone containing either the missing 3' end of clone 3 or the 5' end of clone 4 is not available in the cDNA library.3' RACE (Rapid Amplification of cDNA Ends) was used to amplify and isolate the missing 3' end of clone 3. Reverse transcriptase-polymerase chain reaction (RT-PCR) using a gene-specific primer resulted in a single band of 800 bp representing the 3' end of clone 3.This fragment was cloned and sequenced.In combination with the original clone 3, this sequence completes a cDNA representative of a full-length message for a lectin.The cDNA is 1220 bp long including sequence for the signal peptide and the 3' non-translated region through the polyA tail.The 972 bp open reading frame (ORF) encodes a 324 amino acid polypeptide including a 20 residue signal sequence.The calculated molecular weight of the 304 amino acid protein is 34056 Da.Two potential glycosylation sites are present.Grasshopper lectin is unique among invertebrate lectins having two homologous carbohydrate recognition domains (CRD).This duplication may contribute to the dual carbohydrate specificity (galactose/glucose) shown by this protein.The sequence defining each CRD demonstrates ≈ 30% identity with other vertebrate and invertebrate C-type lectins and 100% identity at 7 invariant amino acid positions within the CRD.5' RACE was used to amplify and isolate the missing 5' end of clone 4. PCR using a clone 4-specific primer resulted in a single band of ≈ 1000 bp which contained the missing 5' end of this clone.This fragment was cloned and partially sequenced.In combination with sequence known for the original clone 4, the new sequence completes a cDNA representative of a full-length message for a second lectin.The complete sequence is 1213 bp long including sequence representing the signal peptide and the 3' non-translated region through the polyA tail.A 978 bp ORF encodes a 326 amino acid polypeptide including a 21 residue signal sequence.The calculated molecular weight of this 305 amino acid protein is 34401 Da.Clone 3 and Clone 4 cDNAs are 81% identical.A 3D homology model of CRD2 from clone 3 was constructed using the crystal structures for rat mannose binding protein (MBP) and E-Selectin (ESEL) as references.Assessment of the model using φψ angles in a Ramachandran plot, dihedral angles and 3D Profiles suggests the model CRD structure is a reasonable 3D representation of the CRD sequence.
Author: Jay R. Radke
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W80038442