KLK1 Human, His

What is Human Kallikrein 1 (KLK1) and what is its primary function?

Human Kallikrein 1 (KLK1), also known as tissue kallikrein, is a serine protease belonging to the S1 serine protease superfamily. It is a member of the human tissue kallikrein family. The primary physiological function of KLK1 is the enzymatic cleavage of low molecular weight kininogen (LMWK) to release vasoactive kinin peptides, specifically lysyl-bradykinin (kallidin) or bradykinin . These kinin peptides regulate numerous physiological processes including blood pressure reduction, vasodilation, smooth muscle relaxation and contraction, pain induction, and inflammation. Additionally, KLK1 plays roles in angiogenesis and tumorigenesis processes .

What is the structural composition of human KLK1?

Human KLK1 precursor contains three distinct regions:

• A signal peptide (residues 1 to 18)

• A short pro-peptide (residues 19 to 24)

• A mature chain (residues 25 to 262)

In recombinant protein formulations, KLK1 is often expressed with tags such as C-terminal 6His or 10His tags for purification purposes, and may include propeptides from other kallikreins (e.g., KLK5 propeptide) to stabilize the protein . The mature protein has a molecular mass of approximately 28.2 kDa, though the apparent molecular mass after post-translational modifications is around 38 kDa .

How does KLK1 differ from other members of the kallikrein family?

KLK1 is one of 15 members of the human kallikrein gene family, all located in a gene cluster on chromosome 19q13.4. What distinguishes KLK1 from other family members is its true kininogenase activity. While other kallikreins like KLK2 and KLK3 (prostate-specific antigen or PSA) share structural similarities and chromosomal location with KLK1, they possess either very low (KLK2) or no (KLK3) kininogenase activity . KLK1 is functionally conserved across species in its capacity to cleave low molecular weight kininogen, though its evolutionary relationship with rodent kallikreins shows divergence after the separation of primate and rodent lineages .

What are the optimal storage and handling conditions for recombinant KLK1 protein?

Recombinant human KLK1 protein requires careful handling to maintain activity. Based on commercial specifications:

• Storage temperature: Store at -20°C or below for long-term stability

• Stability: Stable for approximately 6 months when properly stored

• Formulation: Typically supplied in buffer solutions containing TrisHCl, NaCl, and CaCl2 (e.g., 20mM TrisHCl, 150mM NaCl, 2mM CaCl2, pH 8.0)

• Shipping: Usually shipped frozen with blue ice/gel packs

• Handling: Minimize freeze-thaw cycles to preserve enzymatic activity

• Filtration: Solutions are typically 0.2 μm filtered for sterility

How can I verify the enzymatic activity of recombinant KLK1 in laboratory settings?

To verify KLK1 enzymatic activity, fluorogenic peptide substrate assays are commonly employed. A standardized protocol includes:

• Activation step (if using KLK1 with propeptide):

  • Incubate KLK1 with thermolysin (1:10 ratio thermolysin:KLK1) in assay buffer

  • Incubate at 37°C for 1 hour

  • Stop reaction with 20 mM 1,10 Phenanthroline (final concentration 10 mM)

• Activity assay:

  • Dilute activated KLK1 to 0.4 ng/μL in assay buffer

  • Prepare fluorogenic substrate at 200 μM in assay buffer

  • Combine 50 μL of diluted KLK1 with 50 μL substrate

  • Include substrate blank (50 μL buffer + 50 μL substrate)

  • Monitor fluorescence at excitation/emission wavelengths of 380 nm/460 nm in kinetic mode

  • Calculate specific activity from the reaction kinetics

What methodological approaches are used to study KLK1 inhibition in experimental settings?

Several methodological approaches are employed to study KLK1 inhibition:

• Human monoclonal antibody inhibition assays:

  • Specific antibodies have been developed that selectively inhibit KLK1

  • These can be used in enzyme assays to determine inhibition constants

  • Surface plasmon resonance (SPR) techniques quantify binding kinetics and affinity

• Serine protease inhibitor studies:

  • Natural inhibitors like SPINK6 (serine protease inhibitor of Kazal-type 6) can be tested

  • Inhibition constants (Ki) determination using concentration-dependent inhibition curves

  • Pre-incubation of KLK1 with potential inhibitors before substrate addition

• Small molecule inhibitor screening:

  • High-throughput fluorogenic substrate assays with potential inhibitor compounds

  • Structure-activity relationship studies based on inhibition efficacy

  • Molecular docking simulations to predict binding modes

How is KLK1 being evaluated as a therapeutic target in cardiovascular and renal disorders?

KLK1 has emerged as a promising therapeutic target, particularly for cardiovascular, cerebrovascular, and renal disorders. Current research approaches include:

• Gene therapy approaches:

  • Delivery of KLK1 gene to target tissues using viral vectors

  • Stem cell-based delivery systems incorporating KLK1 genetic modifications

  • Evaluation of tissue-specific expression systems for localized effects

• Therapeutic mechanisms under investigation:

  • Enhancement of nitric oxide (NO) production via bradykinin receptor activation

  • Promotion of angiogenesis in ischemic tissues

  • Anti-inflammatory effects in vascular and renal tissues

  • Modulation of blood pressure regulation pathways

• Preclinical and clinical evidence:

  • Animal models showing improved outcomes in ischemic conditions

  • Preliminary human studies exploring safety and efficacy

  • Biomarker development to monitor therapeutic effectiveness

What are the current challenges in developing KLK1-based therapeutics?

Several significant challenges exist in developing KLK1-based therapeutics:

• Delivery challenges:

  • Protein stability issues affecting half-life in circulation

  • Targeted delivery to affected tissues to minimize systemic effects

  • Optimization of gene therapy vectors for safe, long-term expression

• Functional concerns:

  • Potential immunogenicity of recombinant or modified KLK1

  • Balancing therapeutic effects with potential pro-inflammatory actions

  • Risk of hypotension from excessive vasodilation

• Regulatory and development challenges:

  • Need for tissue-specific or controllable expression systems

  • Development of appropriate biomarkers to monitor efficacy

  • Integration with existing treatment modalities

• Research methodology limitations:

  • Variations in activity measurement protocols affecting comparability

  • Differences between recombinant and native protein activities

  • Species-specific differences in KLK1 function complicating preclinical studies

How do post-translational modifications affect KLK1 function and how can these be analyzed?

Post-translational modifications (PTMs) significantly impact KLK1 functionality, and analyzing these modifications requires sophisticated approaches:

What factors should be considered when designing experiments to evaluate KLK1's role in angiogenesis and tumor biology?

When investigating KLK1's involvement in angiogenesis and tumor biology, researchers should consider:

• Model system selection:

  • Cell models: Endothelial cells (HUVECs, HMVECs) for angiogenesis studies

  • 3D models: Matrigel tube formation assays, spheroid models

  • In vivo models: Appropriate tumor xenograft or genetic models

  • Patient-derived samples: Primary cultures or tissue sections

• Experimental variables:

  • KLK1 concentration ranges physiologically relevant to target tissues

  • Time-dependent effects (acute vs. chronic exposure)

  • Interaction with extracellular matrix components

  • Cross-talk with other proteases and inhibitors in the microenvironment

• Readout selection:

  • Angiogenesis markers: VEGF expression, CD31 staining, tube formation

  • Tumor progression indicators: Proliferation, invasion, metastasis

  • Signaling pathway activation: Bradykinin receptor signaling, NO production

  • Protease activation cascades: Downstream protease activation

• Control considerations:

  • Use of specific KLK1 inhibitors as negative controls

  • Catalytically inactive KLK1 mutants to distinguish enzymatic from non-enzymatic effects

  • Bradykinin receptor antagonists to identify downstream mediators

How can researchers resolve contradictory data regarding KLK1 effects in different experimental systems?

Resolving contradictory data about KLK1 effects requires systematic approaches:

• Source of contradictions:

  • Species differences in KLK1 function and regulation

  • Variations in experimental conditions (pH, ionic strength, substrate availability)

  • Different cell types or tissues exhibiting context-dependent responses

  • Methodological differences in activity measurement or protein preparation

• Resolution strategies:

  • Head-to-head comparisons using standardized protocols

  • Comprehensive characterization of KLK1 preparations (activity, purity, modifications)

  • Inclusion of multiple readouts to capture diverse biological effects

  • Detailed reporting of experimental conditions to facilitate replication

• Mechanistic investigations:

  • Identification of cell-specific receptor and signaling pathway variations

  • Examination of proteolytic processing differences between systems

  • Analysis of local inhibitor concentrations affecting net activity

  • Investigation of compensatory mechanisms in different models

• Integration approaches:

  • Meta-analysis of published data with attention to methodological variations

  • Development of mathematical models to account for system-specific parameters

  • Collaborative validation across multiple laboratories using shared protocols and reagents

What are the optimal experimental approaches for studying KLK1 interactions with other kallikrein family members?

To effectively study interactions between KLK1 and other kallikrein family members:

• Enzymatic activation cascades:

  • Sequential incubation experiments to detect cross-activation

  • Activity-based protein profiling with selective probes

  • Time-course analysis of activation patterns

  • Mass spectrometry identification of cleavage products

• Protein-protein interaction studies:

  • Co-immunoprecipitation experiments with tagged kallikreins

  • Surface plasmon resonance (SPR) for binding kinetics

  • Proximity ligation assay (PLA) for in situ interaction detection

  • Yeast two-hybrid or mammalian two-hybrid screening

• Co-expression analysis:

  • qPCR for co-expression patterns in tissues

  • Single-cell RNA sequencing to identify co-expressing cells

  • Immunohistochemistry co-localization studies

  • Conditional expression systems to manipulate expression ratios

• Functional consequence investigation:

  • Substrate specificity alterations with kallikrein combinations

  • Changes in inhibitor sensitivity in the presence of multiple kallikreins

  • Altered cellular responses to combined kallikrein actions

  • In vivo phenotypes with multiple kallikrein manipulations

What are common technical challenges when working with recombinant KLK1 and how can they be addressed?

Researchers working with recombinant KLK1 often encounter several technical challenges:

• Activity loss during storage/handling:

  • Solution: Aliquot protein upon receipt to minimize freeze-thaw cycles

  • Add stabilizing agents like glycerol (10-20%) for storage

  • Include calcium in buffers (e.g., 2mM CaCl₂) to maintain structural integrity

  • Monitor activity regularly with standardized assays

• Variable activation efficiency:

  • Solution: Optimize thermolysin:KLK1 ratios empirically for each lot

  • Carefully control incubation time and temperature

  • Use activity assays to confirm complete activation

  • Consider alternative activation methods if inconsistency persists

• Background/non-specific activity:

  • Solution: Include appropriate controls (substrate-only, heat-inactivated enzyme)

  • Use highly specific substrates for KLK1

  • Pre-clear solutions by high-speed centrifugation

  • Test for cross-reactivity with other proteases in your system

• Buffer compatibility issues:

  • Solution: Perform buffer exchange using desalting columns if needed

  • Test activity in final experimental buffers before full experiments

  • Supplement with calcium if using chelating agents

  • Monitor pH stability of your working solutions

How can researchers distinguish between direct KLK1 effects and those mediated through bradykinin receptor signaling?

Distinguishing direct KLK1 effects from bradykinin-mediated effects requires:

• Pharmacological approaches:

  • Use selective bradykinin B1 and B2 receptor antagonists (e.g., HOE-140 for B2)

  • Compare native KLK1 effects with direct bradykinin administration

  • Employ selective inhibitors of downstream bradykinin signaling (NOS inhibitors, cyclooxygenase inhibitors)

  • Use engineered KLK1 mutants with altered kininogenase activity but preserved protein structure

• Genetic approaches:

  • Utilize bradykinin receptor knockout models or cells

  • Employ siRNA/shRNA knockdown of bradykinin receptors

  • Use CRISPR/Cas9 to create receptor-deficient cell lines

  • Compare phenotypes between KLK1 knockout and bradykinin receptor knockout models

• Biochemical strategies:

  • Measure bradykinin production directly with mass spectrometry or immunoassays

  • Use kininogen-depleted systems to eliminate bradykinin generation

  • Employ kininase inhibitors to prevent bradykinin degradation

  • Analyze time courses to separate direct (rapid) vs. bradykinin-mediated (delayed) effects

What analytical methods provide the most accurate assessment of KLK1 enzymatic activity in complex biological samples?

For accurate assessment of KLK1 activity in complex biological samples:

• Selective substrate approaches:

  • Fluorogenic substrates with high selectivity for KLK1

  • Internally quenched FRET peptide substrates for improved signal-to-noise

  • Comparison of cleavage patterns with multiple substrates to confirm specificity

  • Spike-in of known amounts of active KLK1 as internal calibrators

• Immunocapture-activity assays:

  • Capture KLK1 using specific antibodies immobilized on plates or beads

  • Wash to remove interfering substances

  • Add specific substrate to measure activity of captured enzyme

  • Include controls with KLK1-specific inhibitors

• Mass spectrometry approaches:

  • Multiple reaction monitoring (MRM) of specific KLK1 cleavage products

  • MALDI-TOF analysis of substrate cleavage patterns

  • Activity-based protein profiling with KLK1-selective probes

  • Parallel reaction monitoring for simultaneous quantification of multiple kallikreins

• Data analysis considerations:

  • Calculate activity ratios relative to reference samples

  • Perform specificity controls with selective inhibitors

  • Account for endogenous inhibitors in biological matrices

  • Consider enzyme kinetics (Km, Vmax) in different sample matrices

How is KLK1 being investigated in the context of stem cell-based therapies for human diseases?

KLK1 is emerging as a target in stem cell-based therapeutic approaches:

• Genetic modification strategies:

  • Transduction of mesenchymal stem cells with KLK1-expressing vectors

  • Conditional expression systems allowing regulated KLK1 production

  • CRISPR/Cas9 modification of stem cells for stable KLK1 expression

  • Development of inducible expression systems for temporal control

• Therapeutic applications under investigation:

  • Cardiovascular disorders: Improving tissue perfusion and reducing damage

  • Cerebrovascular conditions: Neuroprotection in stroke models

  • Renal disorders: Protection against fibrosis and promoting regeneration

  • Wound healing: Enhancing angiogenesis and tissue repair

• Delivery approaches:

  • Direct implantation of modified stem cells into affected tissues

  • Systemic administration with homing to sites of injury

  • Encapsulation technologies for sustained release

  • Combined approaches with scaffolds for tissue engineering

• Monitoring methodologies:

  • Real-time imaging of cell fate and KLK1 expression

  • Biomarkers of KLK1 activity in target tissues

  • Functional outcome assessments in disease models

  • Long-term safety and efficacy monitoring strategies

What is known about the role of KLK1 in the pathophysiology of inflammatory diseases and potential therapeutic implications?

KLK1's role in inflammatory diseases presents both challenges and opportunities:

• Dual roles in inflammation:

  • Pro-inflammatory effects: Bradykinin production leading to vasodilation, increased vascular permeability, and pain

  • Anti-inflammatory potential: Activation of regulatory pathways, resolution of inflammation in certain contexts

  • Tissue-specific effects varying by local microenvironment

  • Temporal dynamics with different roles in acute versus chronic inflammation

• Disease-specific findings:

  • Airway diseases: Potential involvement in asthma and COPD pathophysiology

  • Inflammatory bowel diseases: Altered expression in affected tissues

  • Neuroinflammatory conditions: Modulation of microglial responses

  • Dermatological disorders: Role in inflammatory skin conditions

• Therapeutic strategies under investigation:

  • Selective inhibition in pro-inflammatory contexts

  • Augmentation in settings where anti-inflammatory effects predominate

  • Targeted delivery to specific tissue microenvironments

  • Temporal modulation based on disease stage

• Biomarker development:

  • KLK1 activity and expression as indicators of disease severity

  • Monitoring KLK1 inhibition as a pharmacodynamic marker

  • Genetic variations in KLK1 affecting disease susceptibility

  • Combining KLK1 measures with other inflammatory markers for improved diagnostics

How do recent advances in understanding KLK1 structure-function relationships inform drug development strategies?

Advances in understanding KLK1 structure-function relationships are driving new drug development approaches:

• Structural insights guiding inhibitor design:

  • Crystal structures revealing unique features of KLK1's active site

  • Identification of specific binding pockets for selective targeting

  • Understanding conformational changes upon substrate binding

  • Comparative analysis with other kallikreins to enhance selectivity

• Rational design approaches:

  • Structure-based virtual screening for novel inhibitor scaffolds

  • Fragment-based drug discovery targeting specific KLK1 domains

  • Peptide-mimetic approaches based on natural substrates and inhibitors

  • Allosteric modulator development targeting regulatory sites

• Antibody-based strategies:

  • Epitope mapping of function-blocking antibodies

  • Single-domain antibody (nanobody) development for enhanced tissue penetration

  • Bi-specific antibodies targeting KLK1 and disease-specific markers

  • Antibody-drug conjugates for targeted delivery

• Alternative modulation strategies:

  • RNA-based therapeutics (siRNA, antisense oligonucleotides)

  • Small molecule modulators of KLK1 expression

  • Post-translational modification regulators affecting KLK1 activity

  • Proteolysis-targeting chimeras (PROTACs) for selective KLK1 degradation

How does human KLK1 differ from its orthologs in other species, and what implications does this have for preclinical research?

Human KLK1 shows important differences from orthologs in other species:

• Structural and functional comparisons:

  Species	Key Differences	Implications for Research
  Mouse	Multiple gene copies vs. single human gene	Potentially confounding compensation mechanisms
  Rat	Different substrate specificity	May affect translational relevance of findings
  Non-human primates	Higher homology to human KLK1	Better translational models for inhibitor testing
  Other mammals	Variable glycosylation patterns	Altered pharmacokinetics of recombinant proteins

• Evolutionary considerations:

  • Human and rodent kallikrein families diverged after separation of primate and rodent lineages

  • Expanded gene family in rodents (up to 20 genes) compared to humans (15 genes)

  • Human KLK2 and KLK3 have no known orthologs in rodents

  • Conservation of key catalytic residues across species despite sequence variations

• Research strategy adaptations:

  • Careful selection of appropriate animal models based on specific research questions

  • Potential need for humanized animal models for certain applications

  • Parallel testing in multiple species to address translational gaps

  • Development of species-specific reagents and assays

How do functional relationships between KLK1 and other proteases in biological cascades differ across experimental systems?

Functional relationships between KLK1 and other proteases vary considerably across systems:

• Species-specific interaction networks:

  • Different complement of potential activators and substrates

  • Variable expression patterns of interacting proteins

  • Altered regulatory mechanisms for activation cascades

  • Different inhibitor profiles affecting net proteolytic activity

• Experimental system considerations:

  • Cell culture systems may lack key components of activation cascades

  • In vitro biochemical assays fail to capture cellular compartmentalization

  • Animal models reflect species-specific networks rather than human biology

  • Patient samples capture disease-specific alterations in protease networks

• Methodological approaches for network mapping:

  • Degradomics approaches to identify comprehensive substrate profiles

  • Interactome analysis using affinity purification-mass spectrometry

  • Functional screening using siRNA/CRISPR libraries

  • Computational modeling of protease cascades with system-specific parameters

• Translational implications:

  • Need for careful validation across multiple experimental systems

  • Potential for unexpected compensatory mechanisms in different models

  • System-specific biomarkers of KLK1 activity

  • Therapeutic targeting strategies accounting for system-specific networks

What insights have genomic and transcriptomic analyses provided about KLK1 evolution and expression regulation across species?

Genomic and transcriptomic studies have revealed important aspects of KLK1:

• Evolutionary insights:

  • The human KLK locus shows high synteny with corresponding regions in other mammals

  • Human kallikrein genes are transcribed in the same direction and share 70-90% sequence homology

  • All kallikrein genes maintain five coding exons with conserved exon-intron splice sites

  • TATA box variants and polyadenylation signals are conserved across species

• Expression regulation mechanisms:

  • Tissue-specific promoter elements controlling expression patterns

  • Hormone-responsive elements in promoter regions (androgens, estrogens)

  • Epigenetic regulation through DNA methylation and histone modifications

  • Alternative splicing patterns affecting function and regulation

• Comparative expression profiling:

  • Species-specific expression patterns in different tissues

  • Developmental regulation differences between species

  • Disease-associated expression changes with variable conservation

  • Sex-specific expression patterns with implications for disease susceptibility

• Regulatory network evolution:

  • Variable conservation of transcription factor binding sites

  • Species-specific microRNA targeting affecting post-transcriptional regulation

  • Genomic rearrangements affecting enhancer-promoter interactions

  • Lineage-specific transposable element insertions creating novel regulatory elements

What are promising future directions for developing KLK1-targeted diagnostics and therapeutics?

Promising future directions for KLK1-related applications include:

• Advanced diagnostic approaches:

  • Development of highly specific KLK1 activity assays for clinical samples

  • Multi-marker panels combining KLK1 with related biomarkers

  • Point-of-care testing for rapid KLK1 activity assessment

  • Imaging agents targeting KLK1 for non-invasive visualization

• Therapeutic strategies:

  • Gene therapy approaches using tissue-specific promoters

  • mRNA-based therapeutics for transient KLK1 modulation

  • Development of bispecific antibodies with enhanced targeting

  • Small molecule allosteric modulators with improved selectivity

• Precision medicine applications:

  • Genetic profiling to identify patients likely to respond to KLK1-targeted therapies

  • Companion diagnostics measuring KLK1 activity to guide treatment

  • Combination approaches targeting multiple kallikrein family members

  • Patient stratification based on KLK1 pathway activation status

• Novel delivery technologies:

  • Nanoparticle formulations for targeted delivery

  • Cell-based delivery systems with controlled release

  • Tissue-specific activation systems for localized effects

  • Extracellular vesicle engineering for KLK1 delivery

What methodological advances are needed to better characterize KLK1 functions in complex biological contexts?

To advance our understanding of KLK1 functions, methodological improvements are needed:

• Advanced imaging technologies:

  • Live-cell imaging of KLK1 activity using selective reporters

  • Super-resolution microscopy to localize KLK1 in cellular compartments

  • Intravital microscopy for in vivo visualization of KLK1 activity

  • Correlative light and electron microscopy for ultrastructural context

• Systems biology approaches:

  • Multi-omics integration (proteomics, transcriptomics, metabolomics)

  • Network analysis of KLK1-dependent processes

  • Agent-based modeling of KLK1 functions in tissue microenvironments

  • Machine learning algorithms to identify patterns in complex datasets

• Advanced genetic models:

  • Conditional and inducible KLK1 knockout/knockin systems

  • Tissue-specific expression models with controllable levels

  • Humanized animal models expressing human KLK1

  • Patient-derived organoids for disease-specific studies

• Improved biochemical tools:

  • Activity-based probes with enhanced selectivity

  • Engineered KLK1 variants with altered properties

  • Photoactivatable inhibitors for spatiotemporal control

  • Domain-specific interaction disruption approaches

How might emerging computational and structural biology approaches enhance our understanding of KLK1 and accelerate inhibitor development?

Emerging computational and structural approaches offer significant potential:

• Advanced computational techniques:

  • Molecular dynamics simulations of KLK1-substrate/inhibitor interactions

  • Machine learning for prediction of KLK1 substrates and inhibitors

  • Systems pharmacology modeling of KLK1 in disease networks

  • Quantum mechanics/molecular mechanics for transition state analysis

• Structural biology advances:

  • Cryo-electron microscopy of KLK1 complexes

  • Time-resolved crystallography to capture enzymatic intermediates

  • NMR studies of KLK1 dynamics and conformational changes

  • Hydrogen-deuterium exchange mass spectrometry for protein dynamics

• Integration of structural and functional data:

  • Structure-based pharmacophore modeling for inhibitor design

  • Correlation of structural features with functional outcomes

  • In silico mutagenesis to predict functional consequences

  • Virtual screening against diverse KLK1 conformational states

• Novel computational drug design strategies:

  • Fragment-based approaches using structural data

  • Deep learning for de novo inhibitor design

  • Molecular docking with explicit water molecules

  • Free energy perturbation calculations for binding affinity prediction