Kallikrein-related peptidases regulate the skin barrier, neural plasticity, vascular homeostasis, and reproductive function through limited proteolysis. Dysregulated KLK expression, activation, or inhibition can remodel the extracellular matrix and receptor signaling and contribute to tumors, inflammation, and neurological diseases.

Keywords: kallikrein-related peptidase; KLK; proteolytic cascade; skin barrier; neuroinflammation; tumor invasion; PAR; MMP; biomarker; protease inhibitor

1 Classification and Molecular Characteristics of Kallikrein-Related Peptidases

1.1 Gene Composition of the KLK Family

The human kallikrein-related peptidase (KLK) family comprises 15 secreted serine proteases, KLK1 through KLK15, whose genes are clustered within the chromosome 19q13 region. Different KLKs share similar gene structures and protease catalytic domains but differ substantially in promoter regulation, hormonal responsiveness, tissue distribution, and substrate selectivity.

1.2 Precursor Structure of KLK Proteins

KLKs are generally synthesized as preproenzymes containing a signal peptide that directs the protein into the secretory pathway, a propeptide that maintains the enzyme in an inactive state, and a mature serine protease domain. Removal of the signal peptide generates a zymogen. Subsequent hydrolysis of the propeptide at a specific site exposes the amino terminus of the mature protease and rearranges the active site, ultimately producing full catalytic activity.

1.3 Serine Protease Catalytic Center

Mature KLKs contain a catalytic triad composed of histidine, aspartate, and serine. The catalytic serine attacks the substrate peptide bond to form an acyl-enzyme intermediate, which is subsequently hydrolyzed to release the cleavage products. Substrate-binding pockets surrounding the active site determine KLK preferences for the P1 residue and adjacent amino acids and provide the principal structural basis for designing selective substrates, inhibitors, and activity probes.

1.4 Substrate Recognition Specificity of KLKs

Most KLKs exhibit varying degrees of trypsin-like activity and preferentially cleave at the carboxyl side of arginine or lysine. KLK3 and KLK7 show more pronounced chymotrypsin-like substrate preferences and more readily recognize hydrophobic or aromatic residues. Recognition of physiological substrates is also influenced by substrate secondary structure, glycosylation, local charge, and protein-complex state. In vivo functions therefore cannot be inferred solely from short-peptide substrate assays.

1.5 Differences Between KLKs and Plasma Kallikrein

KLK1 through KLK15 belong to the tissue kallikrein-related peptidase family, whereas plasma kallikrein is encoded by the KLKB1 gene and mainly participates in contact activation, coagulation, and bradykinin generation. KLK1 can cleave low-molecular-weight kininogen and release lysyl-bradykinin, but not all other KLK members possess classical kinin-releasing activity. The KLK family therefore should not be directly equated with the plasma kallikrein system.

Table 1 Expression and Functional Characteristics of Major KLK Family Members

2 Regulation of KLK Precursor Activation and Proteolytic Cascades

Figure 1. Regulation and functional consequences of KLK activity balance in physiological homeostasis and disease states

2.1 Secretion and Extracellular Activation of KLK Zymogens

After processing in the endoplasmic reticulum and Golgi apparatus, KLKs are generally secreted into the extracellular space as zymogens. The propeptide prevents formation of the mature amino terminus and maintains the active site in an inactive state. Catalytic activity is acquired only after the propeptide is cleaved. Different KLKs have distinct activation sites and upstream proteases and may be activated by other KLKs, trypsin-like proteases, plasmin, or local processing enzymes.

2.2 Autoactivation and Cross-Activation

Some KLKs can undergo autoactivation and can also cleave other KLK zymogens, thereby forming amplifying proteolytic cascades. In the skin, KLK5 can autoactivate and subsequently activate KLK7 and KLK14, which further increase degradation of stratum corneum proteins. Such cascade architecture enables rapid amplification of local proteolysis but also allows dysregulation at a single control point to cause extensive tissue damage.

2.3 Endogenous Protease Inhibitors

KLK activity is restricted by serine protease inhibitors, Kunitz-type inhibitors, serpin-family proteins, α₂-macroglobulin, and other molecules. In the skin, LEKTI is encoded by SPINK5 and inhibits KLK5, KLK7, and KLK14. KLK1 can be regulated by endogenous inhibitors such as Kallistatin. Inhibitor concentration, affinity, and subcellular distribution jointly determine the effective window of proteolysis.

2.4 Regulation by pH and Ionic Conditions

KLK activity is highly sensitive to local pH. The stratum corneum has a pH gradient from near-neutral conditions in its deeper layers to an acidic surface. Acidification reduces the catalytic activity of KLK5 and KLK7 but also weakens their binding to LEKTI and promotes dissociation of the inhibitory complexes. When barrier damage or inflammation raises the surface pH, KLK5 and KLK7 activity can increase. Calcium, zinc, salt concentration, and local buffering capacity also influence enzyme conformation, substrate binding, and the stability of protease-inhibitor complexes.

2.5 Glycosylation and Protein Localization

KLK glycosylation can affect protein folding, secretion efficiency, zymogen activation, and proteolytic stability. The extracellular matrix, cell-surface glycosaminoglycans, and secretory vesicles can restrict KLK diffusion and concentrate proteolysis within specific tissue regions. Disease-associated changes in glycosylation or membrane structure may expand the effective range of KLKs and alter antibody recognition of KLK proteins.

2.6 Termination of KLK Activity

Activated KLKs can be captured by endogenous inhibitors, undergo autodegradation or inactivating cleavage, or be removed through internalization. Some KLKs generate inactive fragments after extensive proteolysis, allowing natural termination of the proteolytic cascade. Measurement of total KLK protein cannot distinguish zymogens, active enzymes, inhibitor complexes, and degradation products and therefore does not directly represent the true catalytic state.

3 Physiological Functions of the KLK Family in the Skin, Nervous, Circulatory, and Reproductive Systems

3.1 KLKs and Stratum Corneum Desquamation

KLK5, KLK7, and KLK14 in the stratum corneum cleave corneodesmosin, desmogleins, and other corneocyte-adhesion structures, allowing aged corneocytes to be released from the skin surface in an orderly manner. Insufficient KLK activity causes excessive keratin accumulation, whereas excessive activity accelerates degradation of corneocyte junctions and disrupts the barrier. Skin homeostasis therefore depends on a dynamic balance between proteases and inhibitors such as LEKTI.

3.2 KLKs and Cutaneous Inflammation

KLK5 can cleave and activate protease-activated receptor 2 (PAR2), inducing inflammatory mediator release, pruritic signaling, and barrier-repair responses. KLKs can also process antimicrobial peptide precursors and cytokines, linking cutaneous proteolysis with innate immunity. SPINK5 loss of function causes persistent KLK hyperactivation and is an important mechanism underlying barrier disruption and inflammation in Netherton syndrome.

3.3 KLK6 and Neural Tissue Homeostasis

KLK6 is expressed in the central nervous system and can process myelin proteins, extracellular matrix components, and substrates associated with neuroinflammation. Moderate KLK6 activity may participate in tissue remodeling and injury repair, whereas sustained elevation may promote myelin degradation, glial activation, and blood-brain barrier injury. Its effects depend on disease stage and the local inhibitory environment.

3.4 KLK8 and Synaptic Plasticity

KLK8, also known as Neuropsin, regulates cell-adhesion molecules, synaptic structures, and neural network plasticity through limited proteolysis. Its activity participates in certain learning, memory, and stress responses, but excessive activation may also cause synaptic instability and abnormal neuronal excitation. KLK8 function should be evaluated in the context of brain region, activity state, and substrate changes.

3.5 KLK1 in Vascular and Renal Regulation

KLK1 cleaves low-molecular-weight kininogen and releases lysyl-bradykinin, which subsequently promotes vasodilation, endothelial nitric oxide production, and renal sodium and water excretion through kinin receptors. Abnormal KLK1-kinin signaling may affect blood pressure, glomerular blood flow, and tissue injury responses, but it is mechanistically distinct from the KLKB1-mediated plasma contact activation system.

3.6 KLK2, KLK3, and Seminal Liquefaction

KLK2 and KLK3 secreted by the prostate enter seminal fluid and participate in processing proteins within the seminal coagulum. KLK3 cleaves seminal vesicle-derived proteins such as Semenogelin and promotes seminal liquefaction, whereas KLK2 can participate in KLK3 zymogen activation. This cascade facilitates sperm release and motility and provides an important biochemical basis for evaluating prostate secretory function.

3.7 KLKs in the Female Reproductive System

KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, and other members can be expressed in the ovaries, endometrium, cervix, and related secretions and are regulated by estrogen, progesterone, and local inflammation. Their proteolytic activity may participate in ovulation, tissue remodeling, embryo implantation, and reproductive-tract defense, although expression differs substantially among physiological stages.

4 Abnormal KLK Expression in Tumorigenesis, Invasion, and Treatment Resistance

4.1 Dysregulated KLK Expression in Tumors

KLK genes can be regulated by androgen receptors, estrogen receptors, epigenetic modifications, inflammatory transcription factors, and gene copy-number changes. Prostate cancer frequently involves abnormalities in KLK2, KLK3, and KLK4, whereas ovarian, breast, pancreatic, and lung cancers may exhibit dysregulation of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, and other family members.

4.2 Extracellular Matrix Degradation and Invasion

Abnormally activated KLKs can directly cleave laminins, fibronectin, collagen-associated proteins, and cell-adhesion molecules and can amplify matrix degradation by activating other proteases. Loss of basement membrane integrity allows tumor cells to form invasive protrusions and enter surrounding tissues and blood vessels more readily.

4.3 Epithelial-Mesenchymal Transition and Migration

KLKs can alter cell adhesion and cytoskeletal organization by cleaving E-cadherin, activating PAR signaling, and regulating integrin-FAK/Src pathways. Sustained proteolytic signaling can increase migration, reduce epithelial polarity, and promote epithelial-mesenchymal transition. The specific outcome depends on the KLK member, substrate composition, and tumor differentiation state.

4.4 Growth Factors and Tumor Cell Survival

KLKs can cleave insulin-like growth factor-binding proteins, extracellular growth factor precursors, or extracellular domains of membrane receptors, thereby altering growth factor availability and receptor signaling. Some KLKs enhance PI3K/AKT, MAPK/ERK, and cell-survival signaling and allow tumor cells to adapt to hypoxia, nutrient deficiency, and matrix detachment.

4.5 Angiogenesis and the Inflammatory Microenvironment

KLK-mediated matrix degradation can release angiogenic factors stored within the extracellular matrix and promote endothelial-cell migration. KLKs can also regulate myeloid cells, fibroblasts, and inflammatory responses through PARs, cytokines, and complement-related substrates, linking proteolysis with tumor angiogenesis and immune suppression.

4.6 Metastasis and Survival of Circulating Tumor Cells

KLKs can promote tumor-cell entry into the circulation by weakening intercellular adhesion, increasing matrix penetration, and altering vascular barriers. Some KLKs may also help tumor cells adapt to suspension, form cellular aggregates, and establish new proteolytic microenvironments in distant tissues, thereby supporting metastatic colonization.

4.7 Treatment Resistance

Abnormal KLK activity can influence sensitivity to chemotherapy or targeted therapy through activation of growth signaling, cleavage of drug targets, formation of matrix barriers, and regulation of DNA damage responses. Protease networks possess substantial compensatory capacity. When one KLK is inhibited, other KLKs, MMPs, or components of the plasmin system may substitute for its function. Resistance studies should therefore evaluate the broader proteolytic network.

4.8 Bidirectional Regulation of Tumors by KLKs

KLKs do not act as tumor promoters in every cancer. Some KLKs can exhibit tumor-suppressive effects by degrading growth factors, restricting cell adhesion, or generating antitumor cleavage products. The same family member may even have opposing functions at different tumor stages. Increased KLK expression therefore cannot be directly equated with increased protease activity or tumor dependence.

5 Interactions of KLKs With PAR, MMP, uPA, and TGF-β Signaling Networks

5.1 KLKs and PAR Signaling

Some KLKs can cleave the extracellular amino termini of PAR1 or PAR2, exposing new tethered ligands and initiating G protein- and β-arrestin-associated signaling. Connections between KLK4, KLK5, KLK6, KLK14, and PAR signaling can influence proliferation, inflammation, barrier function, and migration. Different cleavage sites may produce receptor activation, biased signaling, desensitization, or other distinct outcomes.

5.2 KLKs and MMP Cascades

KLKs can directly or indirectly promote activation of certain MMP zymogens. MMPs can in turn release growth factors, degrade basement membranes, and alter substrate accessibility to KLKs. Positive feedback between these two protease classes can substantially amplify extracellular matrix degradation. Inhibiting KLKs without evaluating MMP activity may therefore underestimate proteolytic compensation in tumors and inflammatory tissues.

5.3 KLKs and the uPA-Plasmin System

After binding uPAR, uPA converts plasminogen into plasmin, which degrades the extracellular matrix and activates other protease zymogens. Some KLKs can regulate uPA, uPAR, plasminogen, or their inhibitors, while plasmin may participate in KLK zymogen activation. KLKs and the fibrinolytic system therefore jointly control the intensity of proteolysis at invasive margins.

5.4 KLKs and TGF-β Activation

TGF-β is generally stored in the extracellular matrix as part of a latent complex. KLKs can release active TGF-β by directly cleaving the latent complex or indirectly activating MMPs and plasmin. Activated TGF-β promotes fibroblast responses, extracellular matrix remodeling, epithelial-mesenchymal transition, and immune suppression.

5.5 KLKs and Growth Factor-Binding Proteins

KLKs can cleave insulin-like growth factor-binding proteins and other extracellular carrier proteins, altering local growth factor release, stability, and receptor binding. Moderate processing may increase growth factor availability, whereas excessive hydrolysis may inactivate growth factors. The same KLK can therefore produce signaling effects in different directions.

5.6 Feedback Regulation of Protease Networks

Activation of PARs, TGF-β, and growth factor receptors can feed back through transcriptional regulation to alter expression of KLKs, MMPs, uPA, and protease inhibitors. Proteolysis is therefore not only an upstream trigger of signaling pathways but is also controlled by downstream transcriptional programs, forming self-amplifying protease-receptor signaling loops in diseased tissues.

6 Biomarker Value of KLKs in Cancer, Skin Disease, and Neurometabolic Disorders

6.1 PSA and Prostate Cancer Biomarker Systems

KLK3 is prostate-specific antigen (PSA). Serum total PSA, free PSA, and different molecular forms can be used for prostate disease risk assessment and treatment monitoring. PSA is organ-associated but not absolutely tumor-specific. Benign prostatic hyperplasia, inflammation, and procedural stimulation can also cause elevation, so interpretation requires consideration of age, prostate volume, imaging, and histopathology.

6.2 KLK2 and Multi-Kallikrein Models

KLK2 is closely associated with prostate tissue and androgen signaling and can be combined with total PSA, free PSA, intact PSA, and other indicators in multiparametric models. Combined testing can reduce misclassification caused by a single PSA threshold and improve risk stratification of clinically significant prostate cancer. Thresholds must be independently established for different assay platforms and populations.

6.3 KLK Biomarkers in Other Tumors

KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, and other family members have potential value for expression or body-fluid testing in ovarian, breast, lung, and gastrointestinal cancers. Different KLKs may correlate with tumor stage, invasion, recurrence, and prognosis, but individual members generally have limited disease specificity and are more suitable as components of combined biomarker panels or tissue-classification systems.

6.4 KLK Activity Biomarkers in Skin Diseases

Netherton syndrome, atopic dermatitis, rosacea, and certain keratinization disorders may involve increased activity of KLK5, KLK7, or KLK14. Compared with total protein concentration, active KLK levels, LEKTI levels, and specific substrate-cleavage products in the stratum corneum or skin secretions more accurately reflect cutaneous proteolytic imbalance.

6.5 KLK6 and KLK8 in Neurological Diseases

Changes in KLK6 and KLK8 in cerebrospinal fluid, blood, or brain tissue can be used to study demyelination, neuroinflammation, cognitive impairment, and neurodegenerative processes. Because KLK levels are influenced by neural injury, inflammation, and blood-brain barrier disruption, their disease specificity is limited and they should be interpreted together with NfL, GFAP, imaging, and clinical phenotypes.

6.6 KLK1 in Cardiovascular and Renal Diseases

KLK1 in urine, plasma, or tissue can reflect renal secretory function and the state of local kinin signaling. Changes in KLK1 may be associated with hypertension, diabetic nephropathy, ischemic injury, and endothelial function. Results are influenced by renal function, medications, sodium intake, and sample-processing conditions.

6.7 KLK Networks in Metabolic Diseases

Changes in KLK expression in adipose tissue, the liver, and the pancreas may affect inflammation, insulin signaling, vascular function, and extracellular matrix remodeling. At present, most metabolism-related KLK measurements are more suitable for mechanistic research than for use as independent diagnostic indicators without other metabolic markers.

6.8 Combined Biomarkers and Activity Biomarkers

KLK biomarker evaluation should distinguish gene expression, total protein, zymogen, active enzyme, inhibitor complex, and substrate-cleavage products. Combining KLK protein concentration with enzyme activity, endogenous inhibitors, and disease-specific indicators can reflect the state of the proteolytic network more accurately than a single concentration measurement.

Table 2 Major Biomarker Applications of the KLK Family

7 KLK-Targeted Inhibition, Antibody Drugs, and Clinical Translation Research

7.1 Active-Site Small-Molecule Inhibitors

KLK small-molecule inhibitors can reversibly or irreversibly occupy the active site and prevent substrates from entering the catalytic pocket. Selective design requires exploitation of structural differences among KLKs within the S1 and adjacent binding pockets while limiting cross-inhibition of coagulation factors, trypsin, plasmin, and other serine proteases.

7.2 Peptide and Protein Inhibitors

Substrate-mimetic peptides, cyclic peptides, Kunitz domains, and serpin proteins can achieve high affinity through relatively extensive binding interfaces. These molecules are suitable for blocking extracellular KLKs such as KLK5, KLK7, and KLK14, but stability, tissue penetration, immunogenicity, and local delivery efficiency require further optimization.

7.3 KLK-Targeted Monoclonal Antibodies

Antibodies can bind epitopes near the active site of mature KLKs, allosteric regions, or zymogen-specific structures, thereby blocking enzymatic activity, limiting zymogen activation, or clearing circulating protein. Antibodies generally provide high selectivity but have limited penetration into the stratum corneum, dense tumors, and the central nervous system.

7.4 Blockade of Zymogen Activation

Inhibiting upstream KLKs or masking zymogen activation sites can limit multiple downstream KLKs before the proteolytic cascade begins. This strategy may be more effective than inhibiting a terminal enzyme alone, but upstream KLKs often have multiple physiological substrates. Excessive blockade of normal barrier renewal, tissue repair, or reproductive function must be avoided.

7.5 Substrate-Activated Prodrugs

Tumor-associated KLKs can be used to cleave drug-masking structures so that cytotoxic compounds, immune agonists, or imaging probes are preferentially released in regions of high KLK activity. PSA- or other KLK-cleavable sequences can improve tissue selectivity, but the cleavage sequence must be shown not to undergo premature hydrolysis by other proteases in plasma or normal tissues.

7.6 KLK-Targeted Antibodies and Delivery Strategies

Prostate-associated antigens such as KLK2 can be used to develop antibody drugs, bispecific molecules, radionuclide conjugates, or cellular therapy targets. These approaches depend on stable tissue expression and cell-surface accessibility. Secreted KLKs may also create a circulating antigen sink that alters antibody pharmacokinetics and tumor exposure.

7.7 Topical Treatment of Skin Disease

Topical inhibitors targeting KLK5, KLK7, and KLK14 can reduce excessive degradation of corneocyte junctions, PAR2 activation, and release of inflammatory mediators. Topical administration reduces systemic exposure, but the formulation must penetrate the stratum corneum and retain activity at an appropriate pH while avoiding complete suppression of normal desquamation and antimicrobial defense.

7.8 Combination Therapy and Safety

KLK inhibition can be combined with interventions targeting MMPs, uPA, PARs, TGF-β, or growth factor pathways to reduce protease-network compensation. Safety evaluation should consider coagulation and fibrinolysis, skin-barrier function, reproduction, neural tissue remodeling, and wound healing. A reduction in tumor-cell viability alone is insufficient to define the therapeutic window.

8 Experimental Evaluation of KLK Protein Expression, Enzymatic Activity, and Interactions

8.1 Detection of KLK Gene and Protein Expression

KLK transcription can be measured by RT-qPCR, digital PCR, and transcriptome sequencing. Protein levels can be evaluated by Western blotting, immunohistochemistry, immunofluorescence, and ELISA. Antibodies should distinguish the target KLK from homologous family members and should, where possible, specify whether they recognize the zymogen, mature enzyme, or degradation fragments.

8.2 Distinguishing Active KLK From Total KLK

Total protein detection does not directly reflect catalytic capacity. Active KLKs can be detected using active-site probes, immunocapture activity assays, or active-enzyme-specific antibodies, and the active fraction can be calculated relative to total protein concentration. Known inhibitors, catalytically inactive mutants, and substrate-free controls should be included to exclude nonspecific signals.

8.3 Fluorescent and Colorimetric Substrate Assays

Fluorescent, FRET-based, or chromogenic substrates containing specific cleavage sequences can be used to continuously monitor KLK reaction rates. Enzyme concentration, substrate concentration, pH, salt concentration, temperature, and reaction time should be controlled, and Michaelis-Menten analysis or inhibition curves can be used to calculate Km, Vmax, kcat, IC₅₀, and Ki.

8.4 Zymogen Activation and Cascade Analysis

KLK cascade experiments should simultaneously detect disappearance of the upstream zymogen, formation of the mature amino terminus, and cleavage of downstream substrates. An increase in fluorescent substrate signal alone does not prove activation of a particular zymogen. N-terminal sequencing, mass spectrometry, activity probes, or selective inhibitors should be used to confirm the actual activated member.

8.5 Protease Interactions and Inhibition Kinetics

Interactions of KLKs with inhibitors, antibodies, or substrates can be evaluated using surface plasmon resonance, biolayer interferometry, isothermal titration calorimetry, and co-immunoprecipitation. Inhibitor studies should distinguish competitive, noncompetitive, slow-binding, and irreversible inhibition and should determine association rate, dissociation rate, and complex stability.

8.6 Physiological Substrates and Degradomics

Physiological KLK substrates can be identified through N-terminomics, quantitative proteomics, substrate phage display, and peptide-library screening. Candidate cleavage sites should subsequently be validated using purified proteins, mutant substrates, and cellular models to avoid misclassifying secondary protein degradation as direct KLK cleavage.

8.7 Cellular Functional Evaluation

Cellular assays can measure the effects of KLKs on proliferation, apoptosis, migration, invasion, barrier resistance, cytokine release, and PAR signaling. Controls should include KLK knockdown or knockout, catalytically inactive mutants, exogenous active enzymes, and selective inhibitors to distinguish scaffold functions from catalytic functions.

8.8 Animal Models and Pharmacodynamic Evaluation

In vivo studies can measure KLK expression, activity-probe signals, substrate-cleavage products, tissue pathology, and disease phenotypes. Because KLK gene composition, substrate preferences, and antibody cross-reactivity may differ among species, clinical candidates require evaluation using appropriate orthologous proteins, surrogate antibodies, or humanized models.

9 Products

9.1 KLK Proteins, Antibodies, Gene-Intervention Products, and Detection Reagents

The KLK family participates in tissue homeostasis through controlled zymogen activation, proteolytic cascades, and endogenous inhibition. KLK dysregulation can directly degrade extracellular substrates and amplify disease signaling through PAR, MMP, uPA, and TGF-β networks.

For more related articles, please see below:

[1] Protease Inhibitors

[2] Three Main Differences Between AEBSF and PMSF Protease Inhibitors

[3] Structural Basis, Transduction Branches, and Research Applications of the TGF-β Signaling Pathway