# Antimicrobial Activity of Human C-Type Lectin Domain Family 3 Member A (CLEC3A)
## 4 Discussion
From this, it can be concluded that, similar to RegIII\(\alpha\), the N-terminal charged segment of CLEC3A suppresses its antimicrobial activity against _S. aureus_. Neither CLEC3A nor its protein domains CLEC3A Ex3 and CLEC3A Ex23 could affect the growth of _MRSA_. _MRSA_ expresses a penicillin-binding protein that causes resistance to almost all 8-lactam antibiotics and other types of common antibiotics [29]. Thus, it is unknown whether the penicillin-binding protein or other components of _MRSA_ are responsible for protecting against CLEC3A and its protein domains. For therapeutic use, a long biostability is beneficial [6]. Bacteria are able to induce degradation of immunoglobulins and complement factors by secreting proteases [30]. Therefore, we tested the susceptibility of CLEC3A and its protein domains to bacterial proteases by incubating the proteins with bacterial supernatant. We could show that there was almost no susceptibility of the proteins in the supernatant of _S. aureus_ and a low susceptibility in the supernatant of _P. aeruginosa_ after 24 h. We also investigated the biostability of the proteins in murine serum. CLEC3A and CLEC3A Ex23 showed no signs of degradation by serum proteases within 24 h. Surprisingly, CLEC3A Ex3 showed strong degradation within just 15 min. Nevertheless, the biostability of CLEC3A and CLEC3A Ex23 in murine serum seems to be superior to that of the CLEC3A-derived peptide Ex12, for which a complete degradation could be shown in murine serum within 24 h [19]. Structural motifs of peptides or proteins can increase susceptibility, as they serve as recognition patterns for proteases, or decrease susceptibility, as they inhibit protease activation [31]. The structural differences between CLEC3A and CLEC3A Ex23 in comparison to CLEC3A Ex3 and CLEC3A-derived AMP Ex12 probably contribute to divergences of their susceptibility to serum proteases. In conclusion, our study has shown that, besides being a precursor of AMPs, CLEC3A and its protein domains exhibit antimicrobial activity against _P. aeruginosa_ and _S. aureus_. Interestingly, the antimicrobial effect of CLEC3A against _P. aeruginosa_ was significantly stronger than the effect of the gold standard AMP LL-37 and the CLEC3A-derived peptide Ex12. In addition, CLEC3A and its protein domains show low sensitivity to bacterial and serum proteases, which is a major advantage for the systemic application of the proteins in contrast to most AMPs. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app14010184/s1](https://www.mdpi.com/article/10.3390/app14010184/s1)]([https://www.mdpi.com/article/10.3390/app14010184/s1](https://www.mdpi.com/article/10.3390/app14010184/s1)), Figure S1: Recombinant proteins. (A) 2 ug CLEC3A. (B) 2 ug CLEC3A Ex3 (Ex3). (C) 2 ug CLEC3A Ex32 (Ex23). (D) 2 ug Matrilin-3 (Matn3) and 2 ug Cartilage oligomeric matrix protein (COMP). (E) 4 ug SARS-CoV-2 open reading frame 8 protein (ORF8); Figure S2: Viable count assays. (A) Antimicrobial activity of LL-37, CLEC3A Ex2 (Ex2), Matrilin-3 (Mtn3 with poly His-Tag), and Cartilage Oligomeric Matrix Protein (COMP with Twin-Strep-Tag8) against _P. aeruginosa_. (B) Antimicrobial activity of LL-37, CLEC3A Ex2 (Ex2), and ORF8 (SARS-CoV-2) (with Twin-Strep-Tag8) against _P. aeruginosa_. (C) Antimicrobial activity of LL-37, CLEC3A Ex2 (Ex2), and Matrilin-3 (Mtn3 with poly His-Tag) against _S. aureus_. (D) Antimicrobial activity of LL-37, CLEC3A Ex2 (Ex2), and Cartilage Oligomeric Matrix Protein (COMP with Twin-Strep-Tag8) against _S. aureus_; Figure S3: Incubation of CLEC3A, CLEC3A Ex23 (Ex23) and, CLEC3A Ex3 (Ex3) with _P. aeruginosa_, _S. aureus_ and _MRSA_. Full-sized immunoblots; Figure S4.A: Incubation of CLEC3A, CLEC3A Ex23 (Ex23) and, CLEC3A Ex3 (Ex3) with supernatants of _P. aeruginosa_ and _S. aureus_ for different periods. Secondary antibody-only (Controls); Figure S4.B: Incubation of CLEC3A, CLEC3A Ex23 (Ex23) and, CLEC3A Ex3 (Ex3) with supernatants of _P. aeruginosa_ and _S. aureus_ for different periods.