# Design of Histatin 5 Variants for Improved Proteolytic Stability

> **NIH NIH R03** · UNIV OF MARYLAND, COLLEGE PARK · 2020 · $150,592

## Abstract

PROJECT SUMMARY
Candida albicans is an oral commensal organism and an opportunistic pathogen. C. albicans is a frequent
cause of oral infection in patients with compromised immune systems, including AIDS patients and patients
undergoing chemotherapy, and in patients who produce low levels of saliva. To help prevent the burden of C.
albicans from becoming too high, healthy individuals produce several immune molecules in their saliva. One of
the most important of these molecules is histatin 5 (Hst-5), a member of the histatin family of histidine-rich
antimicrobial peptides. Hst-5 is a 24-amino acid peptide and has the strongest candidacidal activity of the
histatin peptides. Although salivary histatin is effective at killing C. albicans cells in the oral cavity, the
pathogen also has a mechanism for evading killing by Hst-5 and other peptides. C. albicans produces a family
of secreted and cell-wall anchored proteases called secreted aspartic proteases (Saps) that are capable of
degrading Hst-5 and making it inactive. Previous work showed that Sap enzymes cleave Hst-5 at lysine
residues, which is also observed in cleavage by C. albicans cells. Additionally, human saliva contains human
and microbial proteases that can also degrade Hst-5. To reduce proteolytic cleavage of Hst-5 and improving its
potential as a therapeutic in the oral environment, we designed several Hst-5 variants with the lysine residues
substituted with a leucine or arginine residue. Initial proteolysis testing showed variants with resistance to
cleavage by Sap2 and Sap9 enzymes and variants with amplified cleavage, highlighting the complexity of the
interaction of the Saps with Hst-5. The modifications in the variants shifted the location of cleavage by the Sap
enzymes and altered the antifungal activity of the degraded peptides. Importantly, peptides with improved
resistance to cleavage maintained significant antifungal activity following incubation with the Sap enzymes.
The substitutions generally did not result in loss of antifungal activity for the intact peptide, and several
peptides exhibited stronger activity. To better understand the complex interaction between the Hst-5 and
proteolytic enzymes in the oral cavity and enable design of improved peptide therapeutics, we propose to
complete two aims in this work. The first aim is to characterize the interaction of salivary proteases and
additional Saps with Hst-5 variants to understand the structure-function relationships that lead to cleavage (and
lack of cleavage) and antifungal activity. The second aim is to use the knowledge gained through our
preliminary data and Aim 1 to design and test a second generation of Hst-5 variants that further explore the
effect of peptide sequence on proteolytic susceptibility. This project will improve our understanding of the
interaction of Hst-5 with C. albicans Sap enzymes and enzymes in human saliva, providing the necessary
knowledge to design peptides that could function as therapeutics t...

## Key facts

- **NIH application ID:** 9875778
- **Project number:** 1R03DE029270-01
- **Recipient organization:** UNIV OF MARYLAND, COLLEGE PARK
- **Principal Investigator:** Amy J Karlsson
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $150,592
- **Award type:** 1
- **Project period:** 2019-12-01 → 2021-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9875778

## Citation

> US National Institutes of Health, RePORTER application 9875778, Design of Histatin 5 Variants for Improved Proteolytic Stability (1R03DE029270-01). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9875778. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*