Identification of serum biomarkers for canine B-cell lymphoma by use of surface-enhanced laser desorption-ionization time-of-flight mass spectrometry.

OBJECTIVE: To identify biomarker proteins for B-cell lymphoma in canine serum by use of surface-enhanced laser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometry and build classification trees with multiple biomarkers that have high sensitivity and specificity for that tumor type. SAMPLE POPULATION: Sera from 29 dogs with B-cell lymphoma and 87 control dogs (approx equal numbers of healthy dogs, dogs with malignant cancers other than B-cell lymphoma, and dogs with various nonneoplastic diseases or conditions). PROCEDURES: Serum samples were fractionated chromatographically and analyzed via SELDI-TOF mass spectrometry. Peak amplitudes of the spectra from the 2 sample groups were compared to identify potential biomarker peaks, and classification trees were built by use of computer software to detect patterns formed by multiple biomarkers among SELDI data sets. RESULTS: Several biomarker protein peaks in canine serum were identified, and a classification tree was built on the basis of 3 biomarker protein peaks. With 10-fold cross-validation of the sample set, the best individual serum biomarker peak had 75% sensitivity and 86% specificity and the classification tree had 97% sensitivity and 91% specificity for the classification of B-cell lymphoma. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of biomarker proteins identified in canine serum, classification trees were constructed, which may be useful for the development of a diagnostic test for B-cell lymphoma in dogs. Further investigation is needed to determine whether these biomarkers are useful for screening susceptible dog populations or for monitoring disease status during treatment and remission of B-cell lymphoma in dogs.

Insect allantoinase: cDNA cloning, purification, and characterization of the native protein from the cat flea, Ctenocephalides felis.

Allantoinase catalyses the hydrolysis of allantoin to allantoic acid. This reaction is a step in the purine degradation pathway, which produces nitrogenous waste for excretion. A cDNA encoding full-length allantoinase was cloned from a Ctenocephalides felis hindgut and Malpighian tubule (HMT) cDNA library. The cDNA encoded a 483 amino acid protein that had 43% identity with the bullfrog Rana catesbeiana allantoinase and contained the conserved histidine and aspartic acid residues required for zinc-binding and catalytic activity. Unlike the bullfrog allantoinase, the C. felis allantoinase sequence was predicted to contain a 22 amino acid signal sequence, which targets the protein to the secretory pathway. Expression of the mRNA was detected by Northern blot in the first, third, and wandering larval stages as well as in fed and unfed adults, but was not seen in eggs or pupae. In adults, mRNA encoding allantoinase was detected only in the HMT tissues. Immunohistochemistry performed using affinity-purified rabbit immune serum generated against purified recombinant flea allantoinase showed that the native protein localized to the HMT tissues in adult fleas. The anti-allantoinase serum recognized two proteins in an adult flea soluble protein extract, one migrating at 56 kDa and the other at 53 kDa. The two proteins were separated by gel filtration chromatography and were both associated with allantoinase activity. The difference in size appeared to be due to a difference in glycosylation of the proteins. The 53 kDa protein was further purified to near homogeneity by affinity chromatography and retained allantoinase activity. A comparison of the sizes of the native and recombinant C. felis proteins indicated that the 53 kDa native protein may be the product of a post-translational cleavage event, possibly at the putative 22 amino acid signal sequence at the N-terminus of the protein.

Cloning and characterization of five cDNAs encoding peritrophin-A domains from the cat flea, Ctenocephalides felis.

Five cDNAs encoding peritrophin-A domains were identified as expressed sequence tags (ESTs) from flea hindgut and Malpighian tubule (HMT) cDNA libraries. The full-length cDNAs for each were subsequently isolated and sequenced. Three of the encoded proteins were similar to published peritrophin sequences, and thus were called "peritrophin-like", or PL1, PL2, and PL3. The other two sequences had similarity to both mucin and peritrophin proteins, and were called "mucin/peritrophin-like", or MPL1 and MPL2. The predicted protein sequences encoded by these cDNAs all contained a signal sequence and one or more peritrophin-A domains, which have been shown in other proteins to bind chitin. Aside from the peritrophin-A domains, the sequences shared little or no similarity to each other or to other proteins in the GenBank non-redundant database. The predicted protein sequences were variable in size, ranging in length from 81 to 453 amino acids. The two MPL proteins contained putative N-linked and O-linked glycosylation sites, including a region of seven nearly perfect tandem repeats in the MPL1 protein sequence. Northern blot analysis of different flea lifestages and fed adult timepoints showed distinct mRNA expression patterns for each gene, although all five transcripts were primarily or exclusively detected in the HMT tissues in adults. The PL1 protein was detected by immuno-blot in soluble and insoluble protein extracts from unfed and fed adult fleas. The PL1 protein from the insoluble fractions appeared to be approximately 1 kDa larger than the PL1 protein from the soluble protein fractions. Immunohistochemistry performed on flea thin sections revealed that the PL1 protein was detected in the Malpighian tubules, hindgut, rectum, and trachea. Unpurified native PL1 protein from both soluble and insoluble protein fractions was tested for chitin-binding activity but did not bind to chitin under the conditions tested. These results show that the flea peritrophin-like proteins may have biological functions that are distinct from the peritrophic matrix and from the binding of chitin.

Identification, characterization, and cloning of an immunoglobulin degrading enzyme in the cat flea, Ctenocephalides felis.

The degradation of cat immunoglobulin G (IgG) in blood-fed adult C. felis midguts was examined. SDS-PAGE analysis of dissected midgut extracts obtained from C. felis that had been blood fed for various times between 0 to 44 h revealed that by 24 h most of the high molecular weight proteins, including the heavy chain of IgG, were digested. A 31-kDa serine protease with IgG degrading activity was purified from fed C. felis midguts by benzamidine affinity chromatography, hydrophobic interaction chromatography, and cation exchange chromatography. Three primary cleavage products between 30- and 40-kDa were observed when the purified protease was incubated with protein A purified cat IgG. N-terminal amino acid sequence analysis of the products revealed that the IgG degrading protease cleaves after specific cysteine and lysine residues within the hinge region of IgG. The enzyme is also capable of degrading other immunoglobulins, serum albumin, and hemoglobin, suggesting that it may have roles in both combating the host's immune system and providing nutrients for the flea. A cDNA clone encoding the 265 amino acid IgG degrading protease proenzyme was isolated. When expressed in a baculovirus/insect cell expression system, the recombinant protein had the same N-terminus as the processed 237 amino acid mature native protein and possessed IgG degrading activity indistinguishable from the native protein. Arch. Insect Biochem.