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Influenza virus hemagglutinin is a homotrimeric spike glycoprotein crucial for virions' attachment, membrane fusion, and assembly reactions. X-ray crystallography data are available for hemagglutinin ectodomains of various types/subtypes but not for anchoring segments. To get structural information for the linker and transmembrane regions of hemagglutinin, influenza A (H1-H16 subtypes except H8 and H15) and B viruses were digested with bromelain or subtilisin Carlsberg, either within virions or in non-ionic detergent micelles. Proteolytical fragments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Within virions, hemagglutinins of most influenza A/Group-1 and type B virus strains were more susceptible to digestion with bromelain and/or subtilisin compared to A/Group-2 hemagglutinins. The cleavage sites were always located in the hemagglutinin linker sequence. In detergent, 1) bromelain cleaved hemagglutinin of every influenza A subtype in the linker region; 2) subtilisin cleaved Group-2 hemagglutinins in the linker region; 3) subtilisin cleaved Group-1 hemagglutinins in the transmembrane region; 4) both enzymes cleaved influenza B virus hemagglutinin in the transmembrane region. We propose that the A/Group-2 hemagglutinin linker and/or transmembrane regions are more tightly associated within trimers than type A/Group-1 and particularly type B ones. This hypothesis is underpinned by spatial trimeric structure modeling performed for transmembrane regions of both Group-1 and Group-2 hemagglutinin representatives. Differential S-acylation of the hemagglutinin C-terminal anchoring segment with palmitate/stearate residues possibly contributes to fine tuning of transmembrane trimer packing and stabilization since decreased stearate amount correlated with deeper digestion of influenza B and some A/Group-1 hemagglutinins.

Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0-9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.

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