Sociedade Brasileira de Espectrometria de Massas – BrMASS
PROTEOME (PRO)
Saliva, Peptides and Kallikreins (Proteases). How Mass
Spectrometry helped discover its action in Candida albicans.
Douglas Andrade1*, Juliana Rodrigues Oliveira1, Lilian Caroline G. Oliveira1,
Marcia Yuri Kondo1, Jorge Alexandre N. Santos1, Luiz Juliano Neto1, Maria
Aparecida Juliano1
*
1
[email protected]
Instituto Nacional de Farmacologia,
Universidade Federal de São Paulo.
Departamento
de
Biofísica,
Histatins were firstly described by Troxler (1988) [1] as a group of histidine-rich
polypeptides with antimicrobial activity found in human parotid and submandibular
gland secretions. Studies have shown that human salivary antifungal peptide Histatin 5
(H5), product of protease cleavage of Histatin 3 (H3) is taken up by Candida albicans
cells and associates intracellularly with mitochondria [2]. Kallikreins (KLKs) are a group
of 15 secreted serine proteases and some of these KLKs are established or candidates
for cancer biomarkers [3], but the physiological function of most of them remains
unknown. These proteins appear to be highly expressed in one or a few tissues but are
also found at lower concentrations in a wide variety of other tissues [4, 5]. In this work
we investigated the role of Kallikreins 5, 6 and 7 in the proteolytic processing of H3.
These proteases are abundantly found in salivary glands [6, 7] and saliva [5]. We also
determined the catalytic constants (kcat, KM and kcat/KM) for synthetic FRET peptides [8]
(Fluorescence Resonance Energy Transfer) containing sequences of H3. The fragments
of both of peptides, H3 and FRET peptides were analyzed by HPLC (UFLC Proeminence
Shimadzu, Japan), the peaks were collected and indentified by Maldi-Tof/Tof (Autoflex
Speed Bruker Daltonics, Germany). Our results indicate that KLKs 5, 6 and 7 are able
to hydrolyze H3 and FRET peptides with different catalytic efficiency, processing these
substrates in some important antifungal peptides such as H5. It is of great interest,
since the elucidation of salivary protein degradation pathways, so far unknown, can
helps in the development of new pharmaceutical drugs.
References
[1] Diamond, R. D.; Offner, G. D.; Troxler, R. F. J. Biol. Chem. 1988, 263, 7472 - 77.
[2] Helmerhorst, E. J.; Troxler R.F.; Oppenheim F. G. PNAS. 2001, 25, 14637 - 42.
[3] Borgoño, C. A.; Diamandis, E. P. Nature Reviews Cancer. 2004, 4, 876 - 90.
[4] Yousef G. M.; Diamandis E. P. Endocr Rev. 2001, 22, 184 - 204.
[5] Borgoño C. A.; Michael I. P.; Diamandis E. P. Mol Cancer Res. 2004, 2, 257 - 80.
[6] Petraki, C. D.; Papanastasiou P. A.; Karavana, V. N.; Diamandis, E. P. Biol. Chem. 2006,
387, 653 - 663.
[7] Shaw, J. L. V.; Diamandis, Eleftherios P. Clinical Chemistry. 2007, 53, 1423 - 1432.
[8] Korkmaz, B.; Attucci, S.; Juliano, M. A.; Kalupov, T.; Jourdan M. L.; Juliano L.; Gauthier, F.
Nature Protocols, 2008, 3, 1 - 10.
4º Congresso BrMass – 10 a 13 de Dezembro de 2011