Kallistatin is a serine protease inhibitor (serpin) that specifically inhibits tissue kallikrein, a key enzyme involved in kinin generation and vascular homeostasis. While serpins are known to polymerize under certain conditions, the structural properties of human kallistatin—particularly its glycosylation profile, tissue distribution, and polymerization potential—remain poorly defined. In this study, we generated a panel of kallistatin-specific monoclonal antibodies and developed a sensitive sandwich ELISA that enables reliable quantification of kallistatin in both plasma and tissue-conditioned media. The generated antibodies facilitated both quantitative and qualitative analyses, allowing detailed characterization of kallistatin's structural features, glycosylation profile, and localization in vascular tissue from patients with abdominal aortic aneurysm. Using mass-spectrometry, we characterized the glycosylation sites of kallistatin, providing experimental confirmation of the putative glycosylation at Asn 33. Biochemical analyses revealed that kallistatin can polymerize and is susceptible to structural rearrangements typical of serpins. Deglycosylation markedly increased polymer formation demonstrating that glycosylation plays a critical stabilizing role in preventing polymerization. Functional assays using a fluorogenic tissue kallikrein substrate showed that polymerized kallistatin loses inhibitory activity, whereas deglycosylated kallistatin retains normal function. This indicates that glycosylation primarily supports structural stability rather than directly modulating inhibitory capacity. These findings provide new insights into kallistatin's structural features, including its glycosylation, stability, and polymerization behavior, and establish essential tools for further exploring its physiological and pathological roles.
Pham et al. (Mon,) studied this question.