Angiostatin K1-4

What is the molecular structure and organization of Angiostatin K1-4?

Angiostatin K1-4 comprises four kringle modules, each containing two small beta sheets and three disulfide bonds . The K1-3 portion forms a distinct "triangular bowl-like structure" that is stabilized by interactions between inter-kringle peptides and the kringles themselves, although the kringle domains do not directly interact with each other . Functionally, angiostatin is divided into two sides: the K1 side, which is primarily responsible for inhibiting cellular proliferation, and the K2-K3 side, which is primarily responsible for inhibiting cell migration . The K4 domain, present in Angiostatin K1-4 but not in K1-3, affects binding properties with proteins such as tetranectin, which demonstrates significantly higher affinity for K1-4 than for K1-3 .

Through which molecular pathways does Angiostatin K1-4 exert its anti-angiogenic effects?

Angiostatin K1-4, along with other variants, mediates anti-angiogenesis through multiple molecular pathways:

• Upregulation of p53 protein expression and its downstream effectors

• Enhancement of FasL-mediated signaling pathways

• Reduction of AKT activation

• Interaction with at least three different receptors: Fas, integrin alpha(v)beta3, and ATP synthase

• Alteration of approximately 189 genes at the mRNA level, with more than 70% of these genes participating in growth, inflammation, apoptosis, migration, and extracellular matrix functions

These combined mechanisms lead to inhibition of endothelial cell proliferation, migration, and tube formation, as well as induction of apoptosis . Notably, research has identified the involvement of Death-Associated Protein Kinase 1 (DAPK1) in angiostatin-mediated anti-angiogenesis, representing a novel finding in the field .

How does Angiostatin K1-4 interact with the extracellular matrix and why is this significant?

Angiostatin K1-4 binds to the extracellular matrix (ECM) of endothelial cells, which is a novel finding with potential implications for understanding its localization and function in vivo . This ECM binding capability may contribute to the localization of angiostatin at sites where angiogenesis is occurring, potentially enhancing its local anti-angiogenic effects. Research has shown that proteins like tetranectin can significantly reduce the binding of Angiostatin K1-4 to ECM, suggesting a potential mechanism by which tetranectin counteracts angiostatin's anti-angiogenic activity in physiological settings .

How do the anti-angiogenic activities of Angiostatin K1-3, K1-4, and K1-4.5 compare in experimental assays?

Despite mediating anti-angiogenesis through similar molecular pathways, Angiostatin K1-3, K1-4, and K1-4.5 show variations in their efficacy across different experimental assays. Research has demonstrated that these angiostatin molecules at comparable expression levels inhibit various in vitro angiogenesis assays with some differences in potency . The following table summarizes binding affinities of tetranectin variants to different angiostatin forms:

This data shows that the TN T149Y variant has significantly higher binding affinity for AST K1-4 compared to AST K1-3, illustrating the importance of the K4 domain in protein-protein interactions .

What effect does tetranectin have on the anti-angiogenic activity of different angiostatin variants?

Tetranectin significantly counteracts the anti-angiogenic effects of Angiostatin K1-4 on endothelial cell proliferation, as shown in the following experimental results:

Importantly, tetranectin does not have a similar interaction with AST K1-3 or endostatin, indicating that its ability to counteract anti-angiogenic effects is specific to AST K1-4 and likely dependent on binding to the K4 domain .

What are the established methods for producing and purifying Angiostatin K1-4 for research?

Researchers have employed several approaches to produce and purify Angiostatin K1-4:

• Recombinant production systems:

  • Expression in Chinese hamster ovary (CHO) cells, followed by purification procedures

  • Adenoviral expression systems to generate secretable angiostatin molecules for conditioned media collection

  • Pichia pastoris expression systems (though more commonly used for K1-3)

• Natural source isolation:

  • Purification from human plasma, as exemplified by the native AST K1-4 available commercially

Both recombinant and native preparations of angiostatin have demonstrated similar endothelial cell growth inhibitory properties in experimental settings . The choice of production method may depend on specific research requirements and the intended application.

What assays are commonly used to evaluate the anti-angiogenic activity of Angiostatin K1-4?

Multiple in vitro and in vivo assays are employed to comprehensively evaluate the anti-angiogenic properties of Angiostatin K1-4:

• In vitro assays:

  • Endothelial cell proliferation assays

  • Cell migration assays

  • Tube formation assays on extracellular matrix components

  • Apoptosis assays of human endothelial cells

  • Protein binding assays (e.g., to evaluate interactions with tetranectin or other binding partners)

• Molecular assays:

  • Gene expression analysis to identify downstream targets

  • Western blotting to assess changes in protein expression (e.g., p53, AKT)

These complementary approaches allow researchers to assess multiple aspects of Angiostatin K1-4's anti-angiogenic activity and elucidate its underlying mechanisms of action.

How do protein interactions modulate Angiostatin K1-4 activity in physiological settings?

Angiostatin K1-4 activity is modulated by interactions with several proteins:

• Tetranectin: Binds significantly to Angiostatin K1-4 and counteracts its anti-angiogenic effects on endothelial cell proliferation. This interaction appears to be specific, as tetranectin does not similarly counteract the effects of Angiostatin K1-3 or endostatin . The ability of tetranectin to reduce Angiostatin K1-4 binding to endothelial cell ECM suggests one mechanism by which it may promote angiogenesis in vivo.

• β2-glycoprotein I (β2GPI): In its nicked form (cleaved by plasmin in domain V), nicked β2GPI binds to angiostatin 4.5 with high affinity (KD = 3.27 × 10^6 M^−1) . Through this binding, nicked β2GPI attenuates the anti-angiogenic functions of angiostatin 4.5 in proliferation assays, extracellular matrix invasion assays, tube formation assays, and in vivo angiogenesis models . Interestingly, intact β2GPI does not bind to angiostatin 4.5 or inhibit its anti-angiogenic activity.

These interactions highlight the complex regulation of angiostatin activity in vivo and suggest that proteolytic processing of binding partners may be an important mechanism for modulating its anti-angiogenic effects in physiological and pathological settings.

What is the significance of DAPK1 in Angiostatin K1-4-mediated anti-angiogenesis?

The identification of Death-Associated Protein Kinase 1 (DAPK1) involvement in angiostatin-mediated anti-angiogenesis represents a significant finding in the field . DAPK1 is a calcium/calmodulin-dependent serine/threonine kinase that functions as a positive mediator of programmed cell death. Its involvement in the anti-angiogenic effects of angiostatin variants (including K1-4) suggests a mechanistic link between angiostatin's anti-angiogenic activities and apoptotic pathways in endothelial cells.

While the specific mechanisms by which angiostatin activates or regulates DAPK1 were not fully detailed in the available literature, this finding provides an important direction for future research. Understanding the relationship between angiostatin, DAPK1, and endothelial cell apoptosis could potentially reveal new therapeutic targets for anti-angiogenic therapies in cancer and other angiogenesis-dependent diseases.

What are the current challenges and gaps in Angiostatin K1-4 research?

Despite significant advances in understanding Angiostatin K1-4, several challenges and knowledge gaps remain:

• Mechanistic complexity: The molecular mechanism of Angiostatin K1-4 involves multiple pathways and targets, including at least three different receptors (Fas, integrin alpha(v)beta3, and ATP synthase) and effects on numerous genes . Fully elucidating how these pathways integrate to mediate anti-angiogenesis remains challenging.

• Physiological regulation: The interactions with proteins like tetranectin and nicked β2-glycoprotein I suggest complex regulatory mechanisms in vivo . Understanding how these interactions are regulated in different physiological and pathological contexts requires further investigation.

• Structure-function relationships: While the general structural features of Angiostatin K1-4 have been described, more detailed structural studies are needed to understand how specific domains and residues contribute to its anti-angiogenic functions .

• Translational applications: Although angiostatin has shown promise in anticancer therapy , optimizing its delivery, stability, and efficacy for clinical applications remains an area of active research.

Addressing these challenges will require integrated approaches combining structural biology, molecular biology, cell biology, and in vivo models to advance our understanding of Angiostatin K1-4 and its therapeutic potential.

How might gene expression profiling enhance our understanding of Angiostatin K1-4 mechanisms?

Comprehensive gene expression profiling could significantly advance our understanding of Angiostatin K1-4's mechanisms of action. Research has already identified that angiostatin variants alter the expression of approximately 189 genes at the mRNA level, with more than 70% of these genes participating in processes related to growth, inflammation, apoptosis, migration, and extracellular matrix functions .

Future research employing modern transcriptomic approaches (RNA-seq, single-cell RNA-seq) could:

• Identify cell type-specific responses to Angiostatin K1-4 treatment

• Compare the transcriptional effects of different angiostatin variants (K1-3, K1-4, K1-4.5) to better understand their functional differences

• Elucidate the temporal dynamics of gene expression changes following Angiostatin K1-4 treatment

• Identify key transcriptional regulators that mediate Angiostatin K1-4's effects