The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids

Yousef, George M.; Kapadia, Carl; Polymeris, Mary-Ellen; Borgoño, Carla A.; Hutchinson, Shirley; Wasney, Gregory A.; Soosaipillai, Antoninus; Diamandis, Eleftherios P.

doi:10.1016/s0167-4781(03)00116-7

Cited by 30 publications

(13 citation statements)

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“…KLK9 signal was very low in the sera of healthy females and males, in accordance with previous studies [16]. In light of the recovery experiments, we speculated that these findings could point to a significant binding of KLK9 by endogenous serum inhibitors [22–24]. The formation of these heterocomplexes [25] could effectively mask KLK9 epitopes.…”

Section: Discussionsupporting

confidence: 88%

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A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

et al. 2017

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BackgroundKallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples.MethodsThe coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology.ResultsAnti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples.ConclusionsThe ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.Electronic supplementary materialThe online version of this article (doi:10.1186/s12014-017-9140-6) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 88%

“…The formation of these heterocomplexes [25] could effectively mask KLK9 epitopes. This has been observed with many other kallikreins, including PSA [24, 26, 27]. …”

Section: Discussionsupporting

confidence: 60%

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

et al. 2017

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“…5). It is known that serine protease inhibitors in serum, among them ␣ 2 -macroglobulin, bind kallikreins such as hK2, hK3, hK5, and hK13 (41)(42)(43)(44)(45); therefore, hK4 in serum likely forms a complex with ␣ 2 -macroglobulin. This notion is supported by the fact that ␣ 2 -macroglobulin binds many other kallikreins (hK2, hK3, and others) and because of its large size, renders them immunologically undetectable (46,47 ) unless the complex is exposed to a high (nonphysiologic) pH before assaying (48 ).…”

Section: Discussionmentioning

confidence: 99%

Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids

et al. 2005

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“…However, recent studies indicate that AAT is an irreversible inhibitor of other extracellular serine proteases, such as trypsin and kallikreins (5) and matriptase (31)(32)(33). Interestingly, AAT that was taken up by cells can inhibit apoptotic cell death via inhibition of caspase 3 (34).…”

Section: Discussionmentioning

confidence: 99%

Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend”

Shapira

Khalfin

Lewis

et al. 2014

Mol Med

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Autophagy is involved in both the cell protective and the cell death process but its mechanism is largely unknown. The present work unravels a novel intracellular mechanism by which the serpin α 1 -antitrypsin (AAT) acts as a novel negative regulator of autophagic cell death. For the first time, the role of intracellularly synthesized AAT, other than in liver cells, is demonstrated. Autophagic cell death was induced by N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tamoxifen. By utilizing a fluorescently tagged TPCK analog, AAT was "fished out" (pulled out) as a TPCK intracellular protein target. The interaction was further verified by competition binding experiments. Both inducers caused downregulation of AAT expression associated with activation of trypsin-like proteases. Furthermore, silencing AAT by siRNA induced autophagic cell death. Moreover, AAT administration to cultured cells prevented autophagic cell death. This new mechanism could have implications in the treatment of diseases by the regulation of AAT levels in which autophagy has a detrimental function. Furthermore, the results imply that the high synthesis of endogenous AAT by cancer cells could provide a novel resistance mechanism of cancer against autophagic cell death.

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The human kallikrein protein 5 (hK5) is enzymatically active, glycosylated and forms complexes with two protease inhibitors in ovarian cancer fluids

Cited by 30 publications

References 29 publications

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9

Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids

Regulation of Autophagy by α1-Antitrypsin: “A Foe of a Foe Is a Friend”

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