Identification and Characterization of a Sialidase Released by the Salivary Gland of the Hematophagous Insect Triatoma infestans

Abstract

Sialidases (EC 3.2.1.18) are commonly found in viruses, bacteria, fungi, protozoa, and vertebrates, but not in invertebrates. We have previously reported the presence of a new sialidase activity in the gut of exclusively hematophagous insects of the Triatoma genus, which transmit Chagas' disease (Amino, R., Acosta, A., Morita, O. M., Chioccola, V. L. P., and Schenkman, S. (1995) Glycobiology 5, 625-631). Here we show that this sialidase is present in the salivary gland of Triatoma infestans, and it is released with the saliva during the insect bite. The sialidase was purified to homogeneity (>5000 times) to a specific activity of more than 20 units/mg. It elutes from a gel filtration column with a volume corresponding to the size of 33 kDa, and it migrates as a single 26-kDa band in SDS-polyacrylamide gel electrophoresis, which is unusually smaller when compared with other known sialidases. T. infestans sialidase hydrolyzes preferentially ␣233-linked sialic acids at pH 4 -8, with maximal activity between pH 5.5 and 6.5, which is compatible with the optimal pH of secreted sialidases. The sialidase is competitively inhibited by 2-deoxy-2,3-dehydro-Nacetyl-neuraminic acid (K i ‫؍‬ 0.075 mM) and differently from many sialidases, with exception of Salmonella typhimurium sialidase, it is inhibited competitively by HEPES (K i ‫؍‬ 15 mM). The fact that T. infestans sialidase is released with the saliva and can hydrolyze sialylLewis X blood groups, which are the ligands for selectins, suggests that it might have a role in the blood feeding.

New Insights on the Sialidase Protein Family Revealed by a Phylogenetic Analysis in Metazoa

“…However, sialic acids in Protostomia are always detected at low levels compared with Deuterostomia [6] and thus the action of a generic (glyco-)hydrolase, even if with low efficiency on sialic acid containing substrates, may be sufficient in these organisms and substitute the specific activity of sialidases. Studies on Penaeus japonicus (a crustacean, also known as Kuruma shrimp) [49] and Triatoma infestans (a blood-sucker insect belonging to the Hemiptera order, known as barber bug) [50], [51] have also revealed the presence of two secreted proteins with a molecular weight of about 32 and 26 kDa, respectively, acting as sialidase. Because all sialidases studied so far, as well as the proteins identified in this work, have always a molecular mass greater than 40 kDa, it seems unlikely that these enzymes from Protostomia fold in the typical β-propeller sialidase structure of sialidase.…”

Kinetics of expression of the salivary apyrases in Triatoma infestans

“…Similarly, some activities were distinctly localized to different T. infestans gland pairs (Amino et al, 2001;Barth, 1954). In contrast, the sialidase activity was found in D1 and D2 salivary glands of T. infestans (Amino et al, 1998), although the enzyme played no clear role in blood feeding. Therefore, its non-specific location suggests its involvement in gland physiology rather than feeding.…”

“…Sialidase, protease and pore-forming protein activities have been described in the T. infestans saliva (Amino et al, 1998(Amino et al, , 2001(Amino et al, , 2002 but their relevance to blood feeding was not clear. In addition, apyrase activity initially identified in the saliva of T. infestans (Ribeiro et al, 1998) was further characterized as being associated with five proteins, which belong to the 5 0 -nucleotidase family (Faudry et al, 2004).…”

Uptake and incorporation of sialic acid by the tick Ixodes ricinus

“…Arthropod sialidase activity has been detected only in the gut and SGs of a blood-feeding bug Triatoma infestans to date. This bug excretes sialidase into its saliva during blood feeding (Amino et al, 1995(Amino et al, , 1998. Another enzyme, trans-sialidase, is capable of transferring Sia residues to different molecules in the protozoan Trypanosoma cruzi and modifying parasite surface molecules (Pereira et al, 1996).…”