Analyzing the structure of kallikrein-related peptidases with microfluidic modulation spectroscopy

Kallikrein-related peptidases (KLKs) are key markers in cancer regulation, bearing on processes crucial for the survival and spread of cancer cells. For this study, RedShift Bio’s Microfluidic Modulation Spectroscopy (MMS) was used to accurately determine the secondary structural makeup of kallikrein proteins.

MMS results signify that higher-order structure bar charts from MMS are closely correlated with crystal structure expectations, highlighting the role of beta-sheets and turn structures as major components. This consistency emphasizes the similarity among KLK protein types and between KLK and chymotrypsinogen.

Sino Biological’s KLKs exhibit excellent reproducibility in MMS, providing reliable data for drug target investigation. Beyond cancer research, MMS supports an understanding of key interactions while boosting preclinical drug development across various conditions.

KLKs: Key players in cancer regulation

Kallikrein-related peptidases (KLKs), a series of 15 serine proteases (KLK1-KLK15), are crucial in various physiological and pathological processes.^1,2 KLK3, KLK5, KLK6, KLK10, and KLK14 regulate cancer development, invasion, migration, and chemoresistance, affecting molecular networks related to the survival and spread of cancer cells.^1,3–6 

KLKs act as biomarkers and therapy targets in various cancers and can help advance targeted treatments.^7–12 Despite structural similarities, KLKs take on different roles in pathophysiology, underscoring the need to comprehend their structural distinctions, which is key for effective drug design.

Materials and methods

This study used MMS to analyze three KLK proteins: KLK-1, KLK-3, and KLK-7. The sequences of these three KLK proteins show similarity in terms of amino acid residues, as displayed in Table 1, but they vary in terminal cleavage and post-transcriptional modification sites.

Table 1. Sequence similarity between KLK-1, KLK-3, and KLK-7 based on their amino acid residue overlap.¹⁴ Source: RedShift Bio

In opposition to the typical activation near the N-terminus, this study focused on the structure of inactive forms (proenzymes) of these proteins. A comparison of MMS results was cross-referenced with data from chymotrypsinogen, an alpha-chymotrypsin proenzyme similarly activated to KLK proteins.¹³

Sino Biological’s KLK proteins used in this study

Human KLK1 protein (His tag)

HPLC-verified Cat#: 10407-H08H

Purity ≥ 95% as determined by SEC-HPLC. Activity: Measured by its ability to cleave the colorimetric peptide substrate. Image Credit: Sino Biological Inc

Human KLK3/PSA protein (His tag)

HPLC-verified Cat#: 10771-H08H

Purity ≥90% as determined by SEC-HPLC. Activity: Measured by its ability to cleave the colorimetric peptide substrate. Image Credit: Sino Biological Inc

Human KLK7 protein (His tag)

HPLC-verified Cat#: 10416-H08H

>97% as determined by SDS-PAGE. Activity: Measured by its ability to cleave the colorimetric peptide substrate. Image Credit: Sino Biological Inc

The KLK proteins exhibit similar structures: two beta barrels and a prominent unordered structure in crystal formations, as shown in Figure 1. MMS results verify that there is a significant presence of beta-sheet (yellow) and turn structures (light blue) with a comparatively lower alpha-helix (dark blue and purple) content.

Utilizing Visual Molecular Dynamics, MMS can provide a robust, efficient method for analyzing KLK protein conformational structures in a solution. The data enables comparisons to be made and demonstrates key insights into protein activity.¹³ 

Throughout this study, inactive proenzymes were tested, presenting KLK-1's highest activity (>1500 pmoles/min/μg), succeeded by KLK-7 (>150 pmoles/min/μg) and KLK-3 (>100 pmoles/min/μg), contributing to a greater understanding of their specific activities.

Figure 1. Crystal structures of KLK-1 (A)¹⁵, KLK-3 (B)¹⁶, and KLK-7 (C)¹⁷ from the Protein Data Bank. Image Credit: Sino Biological Inc

Results and discussion

Comparing KLK protein types

MMS results are displayed in Figure 2, which demonstrates normalized absolute absorbance spectra (A), second derivative spectra (B), a similarity plot (C), and the Higher Order Structure (HOS) fractional contribution profile (D).

Structural motifs were identified using the Gaussian curve fitting as depicted in the HOS fractional contribution bar chart. Leveraging the overlap area in the inverted and baseline-subtracted second derivative plot, the quantitative results exhibited assessed repeatability against the control sample as shown in Table 2.

Each KLK protein acted as a control (100% similarity), highlighting the nuanced concept of proteins being "differently similar" as a result of their unique structures, in spite of any similarities shared with a third protein.

Figure 2. Absolute Absorbance (A), second derivative (B), Similarity (AO) (C), and HOS bar charts are shown above for KLK-1, KLK-3, and KLK-7. The similarity plot and HOS bar charts detail the differences between the secondary structure components of each type of KLK protein. KLK-1 is green, KLK-3 is pink, and KLK-7 is maroon. Image Credit: RedShift Bio

Table 2. A quantitative analysis of repeatability of each individual measurement as well as the comparison between KLK protein types (the control for each similarity comparison is set at 100%). Source: RedShift Bio

Comparing KLK proteins to chymotrypsinogen

KLK-1 demonstrates dual specificity, exhibiting characteristics of both trypsin and chymotrypsin. On the contrary, both KLK-3 and KLK-7 are predominantly chymotrypsin-like proteins.² This inconsistency, in part, accounts for KLK-1's considerably higher activity (>tenfold) in comparison to the other two KLKs.

During this study, each protein was compared and cross-referenced against chymotrypsinogen MMS results acquired from another study on alpha-chymotrypsin and its proenzyme, chymotrypsinogen.¹⁷ The KLK proteins evaluated here were in their proenzyme (inactive) form, juxtaposed with the inactive form of chymotrypsin, chymotrypsinogen, as shown in Figure 3.

Table 3 outlines the quantitative outcomes of protein similarity via area of overlap analysis of the inverted and baseline-subtracted second derivative plot. Chymotrypsinogen acts as the control, clarifying the similarity of each KLK protein type compared to chymotrypsinogen.

Figure 3. Absolute Absorbance (A), second derivative (B), similarity (C), and HOS bar charts are shown above for KLK-1, KLK-3, and KLK-7. The HOS bar chart details the differences between the secondary structure components of each type of KLK protein from chymotrypsinogen. KLK-1 is green, KLK-3 is pink, KLK-7 is maroon, and chymotrypsinogen is blue. Image Credit: RedShift Bio

Table 3. A quantitative analysis of repeatability of each individual measurement as well as the comparison between KLK protein and chymotrypsinogen (chgen). Source: RedShift Bio

MMS testing demonstrated unique secondary structural components for each KLK protein. KLK-1 and KLK-3 shared the greatest similarity, while KLK-7 distinguished itself. KLK-1 possessed the greatest alpha-helical content, KLK-3 had the most unordered structural contribution, and KLK-7 exhibited the highest beta-sheet content.

Compared to chymotrypsinogen, the KLK proteins were similar but markedly different from one another. Beta-sheets were the main secondary structure in both KLK proteins and chymotrypsin.

Higher-order structure bar charts from MMS were in close correlation with crystal structure expectations, highlighting the fact beta-sheets and turn structures are the main components. This consistency emphasizes the similarity among KLK protein types and between KLK and chymotrypsinogen, as displayed in Figure 4.

Figure 4. Crystal structures of Kallikrein proteins (A) and chymotrypsinogen (B) compared to the relative higher order structure chart (C) for each protein. Green: KLK-1. Pink: KLK-3. Maroon: KLK-7. Blue: Chymotrypsinogen. Image Credit: RedShift Bio

Conclusions

Sino Biological's ultra-pure KLKs play a central role in systematically producing MMS data with outstanding reproducibility. The consistency between MMS findings of Sino Biological's recombinant KLKs and established crystal structures emphasizes the quality and reliability of these proteins.

MMS is an essential tool for investigating the structural composition of non-crystallizable proteins, requiring the highest degree of protein fidelity. Sino Biological's wide variety of recombinant proteins within the same family, which includes KLKs, MMPs, CEACAMs, Cadherins, FcR, and more, allows comparative analyses, which benefits and advances drug target investigations.

Beyond structural analyses, MMS facilitates investigating interactions, such as receptor-ligand and target-antibody, which is essential for preclinical drug development. Sino Biological's wide range of antibodies, which target cancer, neurodegenerative diseases, and other conditions, further illustrates the research potential of this method.

More recombinant KLK proteins from Sino Biological

Source: Sino Biological Inc.

Molecule	Product	Cat#	Purity	Activity
KLK-6	Human KLK6 / Kallikrein 6 / Neurosin Protein (His Tag)	12142-H08H	≥ 95 % as determined by SEC-HPLC
KLK-13	Human KLK13 / Kallikrein-13 Protein (His Tag)	10199-H08H	≥ 95 % as determined by SEC-HPLC	Active
KLK-15	Mouse KLK15 Protein (His Tag)	55382-M08H	> 95 % as determined by SEC-HPLC	Active
KLK-5	Human KLK5 Protein (His Tag)	16006-H08H (Pre-order)	> 90 % as determined by SDS-PAGE.
KLK-4	Human KLK-4 / Kallikrein-4 Protein (His Tag)	11857-H08H	> 94 % as determined by SDS-PAGE	Active
KLK-8	Human KLK-8 / Kallikrein-8 Protein (His Tag)	11820-H08H	> 98 % as determined by SDS-PAGE	Active
KLK-11	Human KLK11 / Kallikrein-11 Protein (His Tag)	10767-H08H	> 90 % as determined by SDS-PAGE	Active
KLK-15	Human KLK15 / Kallikrein-4  Protein (hFc Tag)	12343-H02H	> 80 % as determined by SDS-PAGE
KLK-1	Mouse KLK1 / Kallikrein 1 Protein (His Tag)	50915-M08H	> 90 % as determined by SDS-PAGE	Active
KLK-7	Mouse KLK7 / Kallikrein 7 Protein (His Tag)	50921-M08H	> 95 % as determined by SDS-PAGE	Active
KLK-11	Mouse KLK11 / Kallikrein-11 Protein (His Tag)	50919-M08H	> 95 % as determined by SDS-PAGE

References and further reading

1. Filippou PS, Karagiannis GS, Musrap N, Diamandis EP. Kallikrein-related peptidases (KLKs) and the hallmarks of cancer. Crit Rev Clin Lab Sci. 2016;53(4):277-291. doi:10.3109/10408363.2016.1154643
2. Kalinska M, Meyer-Hoffert U, Kantyka T, Potempa J. Kallikreins - The melting pot of activity and function. Biochimie. 2016;122:270-282. doi:10.1016/j.biochi.2015.09.023
3. Egidi MG, Cochetti G, Serva MR, et al. Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers?. Biomed Res Int. 2013;2013:241780. doi:10.1155/2013/241780
4. Peng Q, Shen Y, Zhao P, Cheng M, Wu Y, Zhu Y. Biomarker implication of kallikrein-related peptidases as prognostic tissue substrates of poor survival in colorectal cancer. Cancer Cell Int. 2020;20:260.doi:10.1186/s12935-020-01350-4
5. Figueroa CD, Molina L, Bhoola KD, Ehrenfeld P. Overview of tissue kallikrein and kallikrein-related peptidases in breast cancer. Biol Chem. 2018;399(9):937-957. doi:10.1515/hsz-2018-0111
6. Gong W, Liu Y, Diamandis EP, et al. Prognostic value of kallikrein-related peptidase 7 (KLK7) mRNA expression in advanced high-grade serous ovarian cancer. J Ovarian Res. 2020;13(1):125. doi:10.1186/s13048-020-00725-5
7. Filippou PS, Karagiannis GS, Musrap N, Diamandis EP. Kallikrein-related peptidases (KLKs) and the hallmarks of cancer. Crit Rev Clin Lab Sci. 2016;53(4):277-291. doi:10.3109/10408363.2016.1154643
8. Hashemipour MA, Fatah FS, Ashraf MJ, Tahmasebi M. Expression of Human Kallikreins 4, 8, 10, 11 and 13 in Pleomorphic Adenomas and Mucoepidermoid Carcinomas. Iran J Pathol. 2016;11(4):334-344.
9. Egidi MG, Cochetti G, Serva MR, et al. Circulating microRNAs and kallikreins before and after radical prostatectomy: are they really prostate cancer markers?. Biomed Res Int. 2013;2013:241780. doi:10.1155/2013/241780
10. Kalinska M, Meyer-Hoffert U, Kantyka T, Potempa J. Kallikreins - The melting pot of activity and function. Biochimie. 2016;122:270-282. doi:10.1016/j.biochi.2015.09.023
11. Zhang L, Lovell S, De Vita E, et al. A KLK6 Activity-Based Probe Reveals a Role for KLK6 Activity in Pancreatic Cancer Cell Invasion. J Am Chem Soc. 2022;144(49):22493-22504. doi:10.1021/jacs.2c07378
12. Bouzid H, Soualmia F, Oikonomopoulou K, et al. Kallikrein-Related Peptidase 6 (KLK6) as a Contributor toward an Aggressive Cancer Cell Phenotype: A Potential Role in Colon Cancer Peritoneal Metastasis. Biomolecules. 2022;12(7):1003. doi:10.3390/biom12071003
13. PDF: How Secondary Structure Relates to Enzyme Activity. https://www.redshiftbio.com/resources/how-secondary-structure-relates-to-enzyme-activity-the-structural-differences-between-a-chymotrypsin-and-its-inactive-precursor-chymotrypsinogen-determined-with-microfluidic-modulation-spectroscopy-mms.
14. UniProt. https://www.uniprot.org/.
15. Laxmikanthan G, Blaber SI, Bernett MJ, Scarisbrick IA, Juliano MA, Blaber M. 1.70 A X-ray structure of human apo kallikrein 1: structural changes upon peptide inhibitor/substrate binding. Proteins. 2005;58(4):802-814. doi:10.1002/prot.20368
16. Ménez R, Michel S, Muller BH, et al. Crystal structure of a ternary complex between human prostate-specific antigen, its substrate acyl intermediate and an activating antibody. J Mol Biol. 2008;376(4):1021-1033. doi:10.1016/j.jmb.2007.11.052
17. Debela M, Hess P, Magdolen V, et al. Chymotryptic specificity determinants in the 1.0 A structure of the zinc-inhibited human tissue kallikrein 7. Proc Natl Acad Sci U S A. 2007;104(41):16086-16091. doi:10.1073/pnas.0707811104

About Sino Biological Inc.

Sino Biological is an international reagent supplier and service provider. The company specializes in recombinant protein production and antibody development. All of Sino Biological's products are independently developed and produced, including recombinant proteins, antibodies and cDNA clones. Sino Biological is the researchers' one-stop technical services shop for the advanced technology platforms they need to make advancements. In addition, Sino Biological offer pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates.

Sino Biological's core business

Sino Biological is committed to providing high-quality recombinant protein and antibody reagents and to being a one-stop technical services shop for life science researchers around the world. All of our products are independently developed and produced. In addition, we offer pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates. Our product quality control indicators meet rigorous requirements for clinical use samples. It takes only a few weeks for us to produce 1 to 30 grams of purified monoclonal antibody from gene sequencing.

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