Dissecting the Cellular Functions of Annexin XI Using Recombinant Human Annexin XI-specific Autoantibodies Cloned by Phage Display

Abstract

Functional studies of cellular proteins are often complicated by the lack of well-defined monoclonal antibodies, the production of which is hampered by the highly conserved nature of these cellular proteins across species. Annexin XI, a member of the Ca 2؉ -dependent, phospholipid-binding protein family, is an example of such a protein and was used in studies to devise a strategy using human autoimmune phage display libraries to generate reagents for biological studies of conserved cellular proteins. An IgG phage display library was generated from bone marrow of an autoimmune patient with high serum antibody titer against annexin XI, which was identified recently as an autoantigen targeted by autoantibodies in several systemic autoimmune diseases. From this phage library, a panel of human monoclonal annexin XI-specific Fabs were isolated and applied to studies of the cellular functions of annexin XI. Confocal microscopy showed a cell cycle-specific redistribution of annexin XI from the cytoplasm to the mitotic spindle. In metaphase, annexin XI was upregulated and costained with ␣-tubulin. The subcellular distribution of annexin XI in COS-7 cells was shown to be Ca 2؉ -dependent, and exhibited a predominantly nuclear pattern at low concentrations and a cytoplasmic pattern at high Ca 2؉ concentrations. Calcyclin, found previously to bind annexin XI in vitro, in vivo coated the nuclear membrane of cultured cell lines and did not colocalize with annexin XI. Ultrastructural analysis by immunoelectron microscopy revealed that annexin XI associated with specific granules in both neutrophils and eosinophils, suggesting a role in the exocytotic pathway. Our results illuminate the multifunctional nature of human annexin XI, provide the first evidence that annexin XI associates with the mitotic spindles and might play a role in cell division, and clearly illustrate the potential of phage display-derived human autoantibodies in broader analyses of the function of highly conserved cellular proteins.

Functional variant ANXA11 R230C: true marker of protection and candidate disease modifier in sarcoidosis

“…8,9 There have been several functions suggested for ANXA11 in calcium signalling, cell division, apoptosis and exocytotic pathway. 10 Nevertheless, the data on the role of ANXA11 in human diseases have been limited; for example elevated ANXA11 protein levels were reported in colorectal cancer tissue correlated with tumour stage and poorer survival. 11 Autoantibodies against ANXA11 (identical to the '56K autoantigen') were identified in patients with autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis and antiphospholipid syndrome.…”

“…However, the wild-type ANXA11 protein and its R230C variant did not differ in terms of sensitivity to Ca2 þ in the recent study. 14 Besides a role in calcium signalling, annexin A11 has been implicated in apoptosis and vesicle trafficking, 8 cell growth, 10 survival of cancer cells 11 and terminal phase of cell division. 15 In this context, the development and maintenance of granulomatous inflammation in sarcoidosis have repeatedly been associated with impaired apoptosis of activated inflammatory cells.…”

Characterisation and protein expression profiling of annexins in colorectal cancer

“…Annexin A11 is implicated as being involved in cell growth (Farnaes and Ditzel, 2003) and a reduction in annexin A11 expression using RNAi stops cell division (Tomas et al, 2004). However, annexin A11 expression was decreased in metastasis, suggesting further dysregulation of this protein with tumour progression and possibly indicating that the tumour microenvironment plays a role in regulating annexin A11, although the specific mechanisms regulating this annexin remain to be elucidated.…”

S100A6 binds to annexin 2 in pancreatic cancer cells and promotes pancreatic cancer cell motility

“…Although we did not study the interaction between tropomyosin and S100A6 here, it is possible that the loss of motility observed after S100A6 depletion is caused, at least in part, by an involvement of S100A6 with tropomyosin. Annexin 11 is widely expressed and roles in cell division (Farnaes and Ditzel, 2003) and cytokenesis (Tomas et al, 2004) have been reported. The binding of annexin 11 to S100A6 has been shown to take place in the nuclear envelope (Tomas and Moss, 2003), where the complex may have a function in membrane deconstruction during nuclear envelope breakdown in mitosis.…”