Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis

Abstract

The saliva of blood-sucking arthropods contains powerful pharmacologically active substances and may be a vaccine target against some vector-borne diseases. Subtractive cloning combined with biochemical approaches was used to discover activities in the salivary glands of the hematophagous fly Lutzomyia longipalpis. Sequences of nine full-length cDNA clones were obtained, five of which are possibly associated with blood-meal acquisition, each having cDNA similarity to: (i) the bed bug Cimex lectularius apyrase, (ii) a 5'-nucleotidase/phosphodiesterase, (iii) a hyaluronidase, (iv) a protein containing a carbohydrate-recognition domain (CRD), and (v) a RGD-containing peptide with no significant matches to known proteins in the BLAST databases. Following these findings, we observed that the salivary apyrase activity of L. longipalpis is indeed similar to that of Cimex apyrase in its metal requirements. The predicted isoelectric point of the putative apyrase matches the value found for Lutzomyia salivary apyrase. A 5'-nucleotidase, as well as hyaluronidase activity, was found in the salivary glands, and the CRD-containing cDNA matches the N-terminal sequence of the HPLC-purified salivary anticlotting protein. A cDNA similar to alpha-amylase was discovered and salivary enzymatic activity demonstrated for the first time in a blood-sucking arthropod. Full-length clones were also found coding for three proteins of unknown function matching, respectively, the N-terminal sequence of an abundant salivary protein, having similarity to the CAP superfamily of proteins and the Drosophila yellow protein. Finally, two partial sequences are reported that match possible housekeeping genes. Subtractive cloning will considerably enhance efforts to unravel the salivary pharmacopeia of blood-sucking arthropods.

Figure 1

Isoelectric focusing gel (negative image) of the salivary apyrase of L. longipalpis, indicating the basic nature of the enzyme. After the run, gels were exposed to 50 mM Tris⋅Cl, pH 8.3/0.1 M NaCl/20 mM CaCl₂/5 mM either ATP or ADP. Reaction was allowed to proceed for 30 min, allowing for calcium phosphate to precipitate, and was stopped by change to the above solution not containing nucleotide. Two pairs of homogenized salivary glands were used per lane.

Figure 2

C-terminal region of the 5′-nucleotidases from Lulo5NUC, A. aegypti apyrase (1703351), and human (112825), mouse (539794) and bovine (461441) 5′-nucleotidase. Note that the insect enzymes do not have the terminal hydrophobic stretch. Lulo5NUC also does not have the consensus serine (*), where the inositol phosphate anchor is predicted to be linked to the mammalian proteins.

Figure 3

Hydrolysis of AMP, ADP, and ATP by salivary gland homogenates of L. longipalpis and its dependence on divalent cations. Reaction medium was one pair of homogenized salivary glands/ml/50 mM Tris⋅Cl/0.1 M NaCl/2 mM nucleotide/2 mM divalent cation, or 0.2 mM EDTA and no divalent cation added. One milliunit = 1 nmol orthophosphate released/min at 37°C. Notice calcium dependence for ATP and ADP hydrolysis but not for AMP. Also note that Mg²⁺ does not substitute for Ca²⁺.

Figure 4

(A) Hyaluronidase activity in L. longipalpis salivary glands. The graph indicates hydrolysis of hyaluronate after incubation with one pair of salivary homogenate per 50 μl reaction mixture. (B) Decrease of salivary hyaluronidase after a blood meal.

Figure 5

(A) Molecular sieving chromatography of 500 pairs of L. longipalpis salivary glands on a TSK-2000SW column perfused with 10 mM Hepes, pH 7.0, and 0.15 M NaCl for 30 min, then a 10-min gradient to 1 M NaCl. Eluted fractions were tested for anticlotting activity on a human plasma recalcification time assay, shown in the symbols. (B) RP fractionation of the most active fractions of the size-exclusion chromatogram on a Hamilton PRP-3 column running a gradient from 10% to 60% acetonitrile in water plus 0.1% trifluoroacetic acid. Symbols indicate the plasma recalcification time with aliquots from the column after drying and reconstitution in 10 mM Hepes, pH 7.0/0.15 M NaCl. The sequence obtained by N-terminal Edman degradation is shown.

Figure 6

(A) Standards of p-nitrophenyl glucosides on RP-HPLC. The numbers refer to the glucoside chain length. The absorbance of the p-nitrophenyl group at 303 nm is shown in the ordinate. (B) Result from incubating one pair of salivary gland homogenate with 0.1 mM p-nitrophenyloctaglucoside at 0- and 90-min incubation. Note conversion of the octaglucoside to the maltoside derivative.