A2LD1 Human

What is A2LD1 and what is its primary function in human biology?

A2LD1, also known as gamma-glutamylaminecyclotransferase (GGACT), is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. This protein demonstrates high activity toward gamma-glutamyl-epsilon-lysine, which derives from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the cyclotransferase fold, a structural feature also observed in gamma-glutamylcyclotransferase enzymes with activity toward gamma-glutamyl-alpha-amino acids. A2LD1 plays a crucial role in the degradation pathway of cross-linked proteins by specifically targeting the cross-links formed between lysine and glutamic acid residues through transglutaminase activity.

What are the known synonyms and alternate nomenclature for the A2LD1 protein?

The A2LD1 protein is known by several synonymous designations in scientific literature and databases. These include:

• Gamma-glutamylaminecyclotransferase (GGACT)

• AIG2-like domain-containing protein 1

• Gamma-glutamylamine cyclotransferase

These alternative names reflect different aspects of the protein's structure, function, or evolutionary relationships, which can be important when conducting comprehensive literature searches or cross-referencing between different databases.

What are the structural characteristics of human A2LD1 protein?

Human A2LD1 protein exhibits distinct structural properties that inform its enzymatic function. The full-length protein spans 153 amino acids and adopts the newly identified cyclotransferase fold. This structural arrangement is shared with gamma-glutamylcyclotransferase, although A2LD1 demonstrates differential substrate specificity. The recombinant form commonly used in research includes an N-terminal His-tag, producing a fusion protein with a molecular weight of approximately 24.1 kDa (217 amino acids total). The protein belongs to the gamma-glutamylcyclotransferase family, which defines its structural classification and evolutionary relationships. The complete amino acid sequence of the recombinant form begins with MGSSHHHHHH SSGLVPRGSH MALVFVYGTL, continuing through the functional domains of the protein.

Which protein family does A2LD1 belong to and what are the implications for its function?

A2LD1 belongs to the gamma-glutamylcyclotransferase family of proteins, which determines many of its biochemical properties and functional mechanisms. This classification reflects the enzyme's ability to catalyze cyclotransferase reactions involving gamma-glutamyl substrates. While other members of this family typically act on gamma-glutamyl-alpha-amino acids, A2LD1 shows specificity toward gamma-glutamyl-epsilon-lysine substrates. The membership in this protein family suggests evolutionary relationships with other cyclotransferases and provides insights into potential structure-function relationships that may be important for experimental design and interpretation. Understanding these family characteristics helps researchers predict potential interaction partners, regulatory mechanisms, and functional redundancies in experimental systems.

What are the optimal storage conditions for maintaining A2LD1 protein stability?

For optimal stability and preservation of enzymatic activity, A2LD1 protein requires specific storage conditions depending on intended duration of storage. For short-term storage (1-2 weeks), the protein can be stored at +4°C in its liquid form. The recommended buffer composition is 20mM Tris-HCl buffer (pH 8.0) containing 1mM DTT and 20% glycerol, which helps maintain protein stability and solubility. For long-term storage, the protein should be aliquoted to minimize freeze-thaw cycles and stored at -20°C to -70°C. It is critically important to avoid repeated freezing and thawing cycles, as these can lead to protein denaturation, aggregation, and loss of enzymatic activity. When preparing working solutions, thaw aliquots rapidly and maintain on ice when not in use to preserve function.

What experimental methods are most suitable for analyzing A2LD1 activity in vitro?

The analysis of A2LD1 enzymatic activity in vitro requires methods that can measure the conversion of gamma-glutamylamines to free amines and 5-oxoproline. Activity assays typically involve monitoring the formation of 5-oxo-L-proline from L-gamma-glutamyl-L-epsilon-lysine substrates. HPLC or LC-MS/MS approaches can quantify reaction products with high sensitivity and specificity. Alternatively, coupled enzymatic assays may be employed where the formation of free lysine is linked to subsequent reactions with spectrophotometric or fluorometric readouts. When designing activity assays, it is important to consider that A2LD1 shows high specificity toward gamma-glutamyl-epsilon-lysine substrates while remaining inactive with L-gamma-glutamyl-alpha-amino acid substrates such as L-gamma-glutamyl-L-alpha-cysteine and L-gamma-glutamyl-L-alpha-alanine. This substrate specificity should inform experimental design and interpretation of results.

How can A2LD1 protein be purified from recombinant expression systems?

Purification of recombinant A2LD1 protein can be achieved through a systematic approach leveraging the His-tag commonly incorporated into expression constructs. The established protocol involves expression in E. coli systems followed by conventional chromatography techniques. For efficient purification, cells are lysed under native conditions, and the clarified lysate is subjected to immobilized metal affinity chromatography (IMAC) using Ni-NTA or similar matrices that bind the N-terminal His-tag. Following elution with imidazole, further purification steps may include size exclusion chromatography to achieve >95% purity as confirmed by SDS-PAGE. The purified protein is typically formulated in 20mM Tris-HCl buffer (pH 8.0) containing 1mM DTT and 20% glycerol at a concentration of approximately 1 mg/ml, as determined by Bradford assay. When designing purification strategies, researchers should consider the potential impact of the N-terminal His-tag on protein folding, activity, and interaction studies.

What is known about the catalytic mechanism of A2LD1?

A2LD1's catalytic mechanism involves the conversion of gamma-glutamylamines to free amines and 5-oxoproline through a cyclotransferase reaction. The enzyme specifically catalyzes the formation of 5-oxo-L-proline from L-gamma-glutamyl-L-epsilon-lysine, contributing to the degradation pathway of proteins cross-linked by transglutaminases. This reaction involves breaking the cross-link between lysine and glutamic acid residues. The specificity of A2LD1 for gamma-glutamyl-epsilon-lysine substrates, rather than gamma-glutamyl-alpha-amino acid substrates, suggests a distinctive binding pocket architecture that accommodates the epsilon-amino group of lysine. The cyclotransferase fold adopted by A2LD1 positions key catalytic residues to facilitate bond cleavage and cyclization, though the specific amino acids involved in catalysis and their precise roles in the reaction mechanism require further structural and biochemical investigation.

How does A2LD1 specificity differ from other gamma-glutamylcyclotransferase family members?

A2LD1 demonstrates a distinctive substrate specificity profile compared to other members of the gamma-glutamylcyclotransferase family. While the family generally catalyzes similar cyclotransferase reactions, A2LD1 exhibits high activity toward gamma-glutamyl-epsilon-lysine, particularly those derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. In contrast, A2LD1 shows negligible activity with L-gamma-glutamyl-alpha-amino acid substrates such as L-gamma-glutamyl-L-alpha-cysteine and L-gamma-glutamyl-L-alpha-alanine, which are typical substrates for other family members. This differential specificity suggests structural adaptations in the substrate binding pocket that accommodate the extended side chain of epsilon-linked lysine residues while disfavoring alpha-linked amino acids. These specificity differences have important implications for the biological roles of these enzymes in different metabolic and catabolic pathways.

What are the implications of A2LD1 activity in protein cross-linking pathways?

A2LD1 plays a significant role in the regulation of protein cross-linking pathways by contributing to the degradation of proteins cross-linked by transglutaminases. Transglutaminases catalyze the formation of isopeptide bonds between glutamine and lysine residues, creating covalent cross-links that increase protein stability and resistance to proteolysis. These cross-links are important in various physiological processes, including blood clotting, wound healing, and extracellular matrix stabilization. A2LD1 specifically degrades the cross-link between lysine and glutamic acid residues, potentially serving as a regulatory mechanism to control the turnover and remodeling of cross-linked proteins. Dysregulation of this pathway could potentially contribute to pathological conditions characterized by abnormal protein cross-linking, such as fibrotic disorders, thrombotic conditions, or neurodegenerative diseases with protein aggregation. Research into A2LD1's role in these contexts represents an important frontier for understanding both normal physiology and disease mechanisms.

How might genetic variants in A2LD1 affect its enzymatic function?

Genetic variants in A2LD1 could significantly impact its enzymatic function through multiple mechanisms. Mutations in the catalytic domain might alter substrate binding affinity, reaction kinetics, or catalytic efficiency. Variants affecting protein stability could reduce steady-state enzyme levels through enhanced degradation or misfolding. Mutations in regulatory regions might alter expression patterns, potentially affecting tissue-specific activity levels. The specificity of A2LD1 for gamma-glutamyl-epsilon-lysine substrates could be modified by variants in substrate recognition domains, potentially broadening or narrowing the range of physiological substrates. While the LOVD database indicates the existence of variants in A2LD1, detailed characterization of their functional impacts remains an open area for investigation. Experimental approaches to address this question could include site-directed mutagenesis followed by in vitro activity assays, structural studies of variant proteins, and cellular studies examining the processing of cross-linked proteins in the presence of A2LD1 variants.

What experimental approaches can be used to study A2LD1 interaction with physiological substrates?

Investigating A2LD1 interactions with physiological substrates requires multifaceted experimental approaches. Pull-down assays using His-tagged recombinant A2LD1 can identify binding partners from cellular lysates, particularly proteins containing gamma-glutamyl-epsilon-lysine cross-links. Surface plasmon resonance or isothermal titration calorimetry can determine binding kinetics and thermodynamic parameters for purified substrate candidates. In vitro enzymatic assays using purified cross-linked proteins can demonstrate substrate conversion, with products identified by mass spectrometry. Cellular approaches might include creating A2LD1 knockout or knockdown models to identify accumulated substrates through comparative proteomics. Fluorescence resonance energy transfer (FRET) constructs could monitor real-time A2LD1-substrate interactions in living cells. Importantly, experimental designs should account for potential physiological substrates beyond the well-characterized gamma-glutamyl-epsilon-lysine derived from fibrin, as A2LD1 might act on diverse cross-linked proteins within different cellular compartments.

What factors might affect A2LD1 activity in experimental settings?

Multiple factors can influence A2LD1 activity in experimental systems, requiring careful consideration during experimental design and interpretation. Buffer composition significantly impacts enzyme function, with optimal activity typically observed in 20mM Tris-HCl (pH 8.0) supplemented with 1mM DTT and 20% glycerol. The presence of reducing agents is particularly important as oxidation of cysteine residues may alter protein folding and catalytic capability. Temperature and pH should be carefully controlled, with deviations potentially altering kinetic parameters or causing protein denaturation. The His-tag present in recombinant forms (MGSSHHHHHH SSGLVPRGSH) may influence activity in some assay formats, particularly those involving conformational changes or protein-protein interactions. Substrate purity and concentration must be optimized for accurate activity measurements. Additionally, potential inhibitors or activators present in complex biological samples could affect activity readings. When inconsistent results are observed, systematic evaluation of these factors should be undertaken to identify and address specific sources of variability.

How can researchers distinguish between A2LD1 activity and other related enzymes in complex biological samples?

Differentiating A2LD1 activity from other related enzymes in complex biological samples presents significant methodological challenges requiring specialized approaches. Substrate specificity offers a primary differentiation strategy - assays employing gamma-glutamyl-epsilon-lysine substrates will preferentially detect A2LD1 activity, while gamma-glutamyl-alpha-amino acid substrates (like L-gamma-glutamyl-L-alpha-cysteine) should show minimal A2LD1-mediated conversion. Specific inhibitors, if available, could selectively block A2LD1 while leaving related enzymes functional. Immunodepletion using A2LD1-specific antibodies prior to activity assays can remove A2LD1 contribution to measured activity. In genetic systems, CRISPR/Cas9-mediated knockout or RNAi-based knockdown can eliminate A2LD1 expression, with remaining activity attributable to other enzymes. For absolute specificity, activity-based protein profiling using substrate analogs that form covalent adducts specifically with A2LD1 could be developed. These approaches can be combined in complementary ways to provide robust discrimination between A2LD1 and related enzymatic activities in complex biological matrices.

What quality control parameters should be evaluated when working with recombinant A2LD1?

Rigorous quality control is essential when working with recombinant A2LD1 to ensure experimental reliability and reproducibility. Purity should be assessed by SDS-PAGE, with commercially available preparations typically exceeding 95% purity. Mass spectrometry confirmation of protein identity and integrity is valuable, particularly to verify the expected molecular weight of approximately 24.1 kDa for the His-tagged construct. Enzymatic activity should be verified using established assays measuring the conversion of gamma-glutamyl-epsilon-lysine to 5-oxoproline. Stability assessment after storage and handling is critical, as protein aggregation or denaturation can significantly impact functional studies. When preparing working solutions, concentration determination by Bradford assay or other protein quantification methods should be performed to ensure accurate dosing in experiments. For long-term studies, batch-to-batch consistency should be evaluated to minimize experimental variability. Maintaining detailed records of these quality control parameters for each protein preparation allows retrospective analysis if unexpected results are observed in subsequent experiments.