SERPING1 Human HEK

What is SERPING1 and what is its primary biological function?

SERPING1, also known as C1 inhibitor (C1INH), C1NH, HAE1, HAE2, or Serpin G1, is a member of the serpin superfamily of serine protease inhibitors. It plays a crucial role in regulating both the complement and contact systems in human physiology. SERPING1 specifically regulates the activation of complement factor C1 and the activity of activated C1 by interacting with the active catalytic sites on the light chains of C1r and C1s .

This inhibitory function prevents uncontrolled activation of the complement cascade, which could otherwise lead to excessive inflammation and tissue damage. SERPING1 deficiency results in hereditary angioedema (HAE), characterized by recurrent episodes of localized angioedema affecting the skin, gastrointestinal mucosa, or upper respiratory tract .

What is the structure and molecular characteristics of recombinant SERPING1 Human HEK?

Recombinant SERPING1 produced in HEK293 cells is a single polypeptide chain containing 486 amino acids (positions 23-500 of the native sequence). The protein is fused to an 8-amino acid His-tag at the C-terminus to facilitate purification .

The molecular structure includes:

• A reactive center loop that acts as a substrate for target proteases

• Multiple glycosylation sites that contribute to stability and function

• The characteristic serpin fold with α-helices and β-sheets

• An 8-amino acid His-tag at the C-terminus for purification purposes

When produced in HEK293 cells, SERPING1 undergoes human-specific post-translational modifications, particularly glycosylation, which are essential for proper folding, stability, and biological activity .

How does recombinant SERPING1 from HEK cells compare to native human SERPING1?

Recombinant SERPING1 produced in HEK293 cells closely resembles native human SERPING1 in several important aspects:

• Post-translational modifications: The HEK293 expression system provides human-like glycosylation patterns critical for proper protein folding and function

• Functional domains: The recombinant protein contains amino acids 23-500, which includes all essential functional domains necessary for serine protease inhibition

• Inhibitory activity: The recombinant protein maintains inhibitory function against target proteases similar to native SERPING1

The primary difference is the addition of the C-terminal 8-amino acid His-tag, which facilitates purification but may slightly alter certain biochemical properties compared to the native protein .

What are the optimal storage and reconstitution conditions for maintaining SERPING1 activity?

To maintain optimal SERPING1 activity, researchers should follow these evidence-based storage and reconstitution guidelines:

Storage Conditions:

• Lyophilized SERPING1 should be stored desiccated below -18°C for long-term stability

• While stable at room temperature for up to 3 weeks in lyophilized form, refrigerated storage is recommended whenever possible

• Avoid exposure to repeated freeze-thaw cycles

Reconstitution Protocol:

• Reconstitute lyophilized SERPING1 in 1× PBS to a concentration no less than 100 μg/ml

• Allow complete dissolution through gentle rotation

• After reconstitution, store at 4°C for short-term use (2-7 days)

• For longer storage, aliquot and store below -18°C

• Prevent freeze-thaw cycles to maintain activity

Following these protocols ensures maximal retention of biological activity for experimental applications.

What experimental approaches can verify SERPING1 activity in research settings?

Researchers can verify SERPING1 activity through multiple complementary approaches:

Biochemical Assays:

• Chromogenic substrate assays to measure inhibition of C1s or plasma kallikrein

• SDS-PAGE analysis to visualize SERPING1-protease complexes under non-reducing conditions

• Enzyme kinetic analyses to determine inhibition constants and association rates

Functional Complement Assays:

• Classical pathway hemolytic assays to assess SERPING1's ability to inhibit complement-mediated lysis

• C4 consumption assays to measure prevention of C4 cleavage

• ELISA-based detection of complement activation markers

Structural Verification:

• Circular dichroism spectroscopy to confirm proper secondary structure

• Size exclusion chromatography to verify monomeric state

A comprehensive verification approach typically includes both direct protease inhibition assessment and functional evaluation in complement regulatory pathways .

How can researchers differentiate between the various functional roles of SERPING1?

To differentiate between SERPING1's diverse functional roles, researchers can employ these methodological strategies:

Pathway-Specific Activation:

• Complement pathway: Use classical pathway activators like immune complexes or C1q

• Contact system: Employ surface activators such as kaolin or ellagic acid

• Coagulation pathway: Utilize tissue factor to initiate the extrinsic pathway

Selective Inhibition Approaches:

• Apply pathway-specific inhibitors alongside SERPING1

• Use blocking antibodies against specific proteases

• Employ selective small molecule inhibitors for target proteases

Kinetic Discrimination:

• Perform time-course experiments to separate rapid reactions from slower ones

• Use pulse-chase approaches to track SERPING1 consumption by different proteases

Pathway-Specific Readouts:

• Measure bradykinin production for contact system activity

• Detect C4a/C3a/C5a generation for complement pathway activation

• Quantify thrombin-antithrombin complexes for coagulation effects

This multi-faceted approach enables researchers to distinguish the specific roles of SERPING1 in different biological systems.

How does SERPING1 specifically interact with complement pathway components?

SERPING1 regulates the complement system primarily at the initiation phase of the classical pathway through a unique "mousetrap" mechanism characteristic of serpins:

• SERPING1 binds to C1r and C1s proteases within the C1 complex, preventing proteolytic activation of downstream complement components

• The interaction involves the reactive center loop (RCL) of SERPING1, which acts as a pseudosubstrate

• Upon RCL cleavage by the protease, SERPING1 undergoes a dramatic conformational change that distorts the active site of the protease

• This results in a stable, irreversible 1:1 stoichiometric complex where both proteins are inactivated

Beyond the classical pathway, SERPING1 also regulates the lectin pathway by inhibiting MASP-1 and MASP-2 (mannan-binding lectin-associated serine proteases), which share structural and functional similarities with C1r and C1s .

Research indicates that the inhibition rate varies between target proteases, suggesting a regulated sequence of inhibitory events during complement activation .

What experimental models best replicate SERPING1 deficiency for studying angioedema pathophysiology?

Several experimental models effectively replicate SERPING1 deficiency for studying angioedema pathophysiology:

Cellular Models:

• HEK293 cells with CRISPR/Cas9-mediated SERPING1 knockout

• Patient-derived fibroblasts or lymphocytes exhibiting SERPING1 deficiency

• Induced pluripotent stem cells from HAE patients differentiated into relevant cell types

Animal Models:

• SERPING1 knockout mice (displaying increased vascular permeability)

• Bradykinin receptor transgenic mice (showing hypersensitivity to bradykinin)

• Factor XII knockout mice (protected from angioedema)

Ex Vivo Systems:

• Human plasma reconstitution assays with purified components

• Endothelial cell monolayer systems to measure barrier function

Each model offers distinct advantages and limitations:

• Cellular models enable detailed molecular studies but lack systemic complexity

• Animal models provide in vivo context but may not fully recapitulate human disease mechanisms

• Ex vivo systems bridge the gap between cellular and animal models but have limited temporal scope

How do mutations in the SERPING1 gene correlate with different types of hereditary angioedema?

Mutations in the SERPING1 gene correlate with different types of hereditary angioedema (HAE) in specific patterns:

HAE Type I (approximately 85% of cases):

• Characterized by low plasma levels of C1-inhibitor protein (quantitative deficiency)

• Associated with nonsense mutations, large deletions/insertions, and frameshift mutations that result in truncated proteins

• These mutations often lead to protein misfolding and retention in the endoplasmic reticulum, triggering degradation

HAE Type II (approximately 15% of cases):

• Characterized by normal or elevated levels of dysfunctional C1-inhibitor (qualitative deficiency)

• Typically associated with missense mutations affecting the reactive center loop or regions critical for conformational change

• Most commonly involves mutations at or near the reactive site (P1-P1' residues)

Table 1: Characteristic SERPING1 Mutation Types and Their Association with HAE Subtypes

The position and nature of SERPING1 mutations provide insights into disease mechanisms and can potentially guide personalized treatment approaches .

How does glycosylation of SERPING1 affect its stability and function in experimental systems?

Glycosylation of SERPING1 significantly impacts its stability and function in experimental systems:

Stability Effects:

• N-linked glycans enhance thermostability and protect against proteolytic degradation

• Glycosylation promotes proper folding during synthesis and secretion

• Deglycosylated SERPING1 shows increased tendency to form polymers and aggregates

• Half-life in circulation is substantially reduced for non-glycosylated variants

Functional Effects:

• Minimal direct impact on protease inhibitory activity against C1s and C1r

• May influence interaction rates with certain proteases like plasma kallikrein

• Affects protein-protein interactions with cellular receptors

In experimental systems, researchers should consider:

• Expression systems (HEK293 cells provide human-like glycosylation patterns)

• Storage conditions (glycosylated SERPING1 is more stable during freeze-thaw cycles)

• Experimental temperature (glycosylation provides greater stability at physiological temperatures)

The recombinant SERPING1 produced in HEK293 cells exhibits glycosylation patterns similar to native human SERPING1, making it suitable for most experimental applications where physiologically relevant function is required.

What considerations are important when designing SERPING1 knockout or knockdown experiments?

When designing SERPING1 knockout or knockdown experiments, researchers should consider these critical factors:

Model Selection:

• Choose cell types with physiological relevance (hepatocytes as producers, endothelial cells as targets)

• Consider species differences in complement and contact systems

• Evaluate primary cells versus cell lines (different baseline expression levels)

Knockout Strategies:

• For CRISPR/Cas9 approaches, design multiple guide RNAs targeting different exons

• Target early exons to ensure complete functional disruption

• Screen for off-target effects

• Consider generating heterozygous models to mimic HAE carrier status

Knockdown Approaches:

• siRNA offers transient effects, useful for acute studies

• shRNA provides stable knockdown for longer experiments

• Include scrambled RNA controls and validate knockdown efficiency

Verification Methods:

• Quantify SERPING1 mRNA levels (qRT-PCR)

• Measure protein expression (Western blot, ELISA)

• Assess functional readouts (protease inhibition assays)

• Confirm phenotypic changes relevant to HAE (e.g., bradykinin production)

A comprehensive experimental design incorporating these considerations will yield more reliable and translatable results.

What experimental controls should be included when studying SERPING1 function in complement regulation?

When studying SERPING1 function in complement regulation, these experimental controls should be included:

Positive Controls:

• Commercial purified human C1-inhibitor (plasma-derived)

• Serum from healthy individuals with normal SERPING1 levels

• Known inhibitors of complement activation (e.g., EDTA for all pathways)

Negative Controls:

• Heat-inactivated SERPING1 (56°C for 30 minutes)

• SERPING1-depleted serum

• Inactive SERPING1 mutant (e.g., reactive center loop mutant)

• Irrelevant protein of similar size and purification method

Specificity Controls:

• Other serpins that do not inhibit complement

• Non-inhibitory fragments of SERPING1

• Titration series to demonstrate dose-dependency

• Pre-incubation with target proteases to demonstrate specificity

System Controls:

• Verification of complement activation in the experimental system

• Temperature controls (4°C vs. 37°C) to distinguish between pathway activation points

• Buffer composition controls (ionic strength, pH, calcium concentration)

These controls help distinguish specific SERPING1 effects from non-specific effects, ensure assay functionality, and provide proper context for interpreting experimental results.