Proteases (also called proteinase or peptidases) are enzymes that catalyse the breakdown of proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. (López-Otín & Bond, 2008) Proteases break the peptide bonds between amino acids by adding a water molecule (hydrolysis) to the amino acid backbone, severing the connecting. Depending on the type of protease, this can have varying results. Endopeptidase can cleave a protein from nonterminal amino acids, resulting in smaller polypeptides. Exopeptidase can only remove terminal amino acids at the end of the protein, resulting in cleavage of individual amino acids.

The nucleus of a protease is a chemically active group. This attacking group is part of an amino acid such as cysteine, serine or aspartate. The other amino acids in the active centre help to catalyse the hydrolysis reaction. There are many different families of proteases. Proteases can be classified into seven broad groups:

• Serine proteases – using a serine alcohol
•  Cysteine proteases – using a cysteine thiol
•  Threonine proteases – using a threonine secondary alcohol
• Aspartic proteases – using an aspartate carboxylic acid
• Glutamic proteases – using a glutamate carboxylic acid
• Metalloproteases – using a metal, usually zinc
•  Asparagine peptide lyases – using an asparagine to perform an elimination reaction (not requiring water) (Oda, 2012)

Protease assays are a real addition to the characterisation of allergenic extracts. Often, allergenic extracts are only defined in the concentration of proteins and their allergenic activity, such as the binding capacity with specific serums. However, this does not paint a full picture of the characterization of an allergen extract. An addition is to test the enzymatic functionality of the extracts. Many allergens have an enzymatic function within its host. Der p 1, a cysteine protease, can be found in the intestinal track and the excrements of Dermatophagoides pteronyssinus. Here, it helps the house dust mite with digesting large particles in the outer environment and converting them into future food. Once the mite eats its excrements again, it can absorb more nutrients (Randall et al, 2017). Measuring the activity of an allergen is a useful addition to the characterisation of this protein.

Cystein protease

The cysteine proteases can be subdivided into several different classes, of which the papain-like protease class is the largest. Multiple major allergens, including Der p 1 and Der f 1, fall into this category. At Citeq, we developed a cysteine protease activity assay to determine activity of proteases of this class, using papain as a standard curve. Papain was the first cysteine protease to be described and is often regarded as the golden standard of cysteine proteases. (Barret, Rawlings, and Woessner, 1998).

Cysteine proteases also have clinical relevance as medicine. The cysteine protease imlifidase has been studied by Jordan et al (2021) as a potential therapy during kidney transplantation to prevent graft rejection by cleaving IgG antibodies, preventing IgG mediated injury to the grafts. On the other hand, cysteine protease SARS-COV-2 pro has been identified as a potential target to treat infection with coronavirus COVID-19 (Arya, 2021). This demonstrate the importance of cysteine proteases in research.

Arya R, Kumari S, Pandey B, Mistry H, Bihani SC, Das A, Prashar V, Gupta GD, Panicker L, Kumar M. Structural insights into SARS-CoV-2 proteins. J Mol Biol. 2021 Jan 22;433(2):166725. doi: 10.1016/j.jmb.2020.11.024. Epub 2020 Nov 24. PMID: 33245961; PMCID: PMC7685130.

Jordan SC, Legendre C, Desai NM, Lorant T, Bengtsson M, Lonze BE, Vo AA, Runström A, Laxmyr L, Sjöholm K, Schiött Å, Sonesson E, Wood K, Winstedt L, Kjellman C, Montgomery RA. Imlifidase Desensitization in Crossmatch-positive, Highly Sensitized Kidney Transplant Recipients: Results of an International Phase 2 Trial (Highdes). Transplantation. 2021 Aug 1;105(8):1808-1817.

Barrett, A. J., Rawlings, N. D. & Woessner, J. F. (1998) Handbook of Proteolytic Enzymes. Academic Press, London

Oda K. (2012) New families of carboxyl peptidases: serine-carboxyl peptidases and glutamic peptidases, the journal of biochemistry,151(1): 13-25