Prevention of proteases by a multifunctional plasma protein: alpha-2 macroglobulin (A2M), can protect us from many diseases.

Mohammad M. Khan, MBBS., PhD. Biomark Bangladesh Foundation, Hosna Centre, Gulshan Avenue, Gulshan, Dhaka 1212, Bangladesh. Author contact: aurnobaunton@gmail.com

Reviewed by Naseem Mahmud, MBBS, PhD, DTM, Obstetrics and Gynaecology, United Hospital, Dhaka, Bangladesh.

INTRODUCTION

Previously we have shown (in online Journal, www.rarediseaseindia.org: www.rarediseasesindia.org/septicshock, 2012)¹ - first time in the world - that alpha-2-macroglobulin (A2M) is the major innate immune factor against foreign protease and exogenous A2M is a major life saving protein in the critical stage of protease induced septic shock animal models^2-5. Recently, in a successful treatment with A2M in osteoarthritis was also shown in human subjects⁶.  Now we are showing the importance of different kind of protease(s) involvement in many common and rare diseases and the treatment with multifunctional broad spectrum protease inhibitor, A2M could be a life saving molecule in the field of medicine^7-8 (Fig.1). We also suggest to include A2M - as a major component in the innate immune system.

PROTEASES

Microorganism(s) invasion, metabolism and virulence are dependent on protease(s) which are secreted by prokaryotic and eukaryotic parasites⁹.  Proteases are secreted by microorganisms to penetrate into the body¹⁰.  There are over 500 human proteases, accounting for 2% of human genes (DNA sequences that code for amino acids), and similar numbers of proteases occur in every plant, insect, marine organism and in all infectious organisms that cause disease¹¹.  Proteases play pivotal regulatory roles in conception, birth, digestion, growth, maturation, ageing, and death of all organisms. Proteases regulate most physiological processes by controlling the activation, synthesis and turnover of proteins. Proteases are also essential in viruses, bacteria and parasites for their replication and the spread of infectious diseases, in all insects, organisms and animals for effective transmission of disease, and in human and animal hosts for the mediation and sustenance of diseases. It is now known that single amino acid mutations in over 50 human proteases result in hereditary/genetic diseases. Also, other genetic or environmental conditions can result in an over- or under- abundance of a particular crucial protease or abnormal levels of natural inhibitors/activators of proteases, leading to abnormal physiology and disease¹². Diseases in which proteases are involved^13-20 are shown in Table 1.

A2M 

A2M, a broad spectrum protease inhibitor, normally is continuously removing proteases, and keep us healthy^7,8,21. But if the protease concentration goes up and A2M is consumed and fail to clear proteases due to less supply to its demand from its original source (liver), then proteases have free access to cleave other proteins of many different systems and start inactivating all of our defense mechanisms. Eventually many toxic molecules are generated and we become sick. At this stage, it is essential to supply more A2M to compensate and remove proteases from body^4,5.

Low level of A2M are well documented indicate a relationship of disproportion of proteases and protease inhibitor in the patho-physiology of many disease processes²². In animal research it was also clearly documented that in a Gram negative bacterial septic shock model, level of A2M went down to 30% of its original concentration and remaining 30% of A2M was not enough to protect animals from death. In that study, injecting purified A2M at the very crisis stage of septic shock was able to rescue animals from death which worked as a life saving medicine^4,5.

A2M, an extraordinary multifunctional blood protein had never been got any attention for a possibility as a life saving therapeutic use in the field of medicine. Therefore, measurement of serum/plasma A2M concentration and different kind of proteases in patients may be a very important indicator for the diagnosis and speculate the severity of disease process. The life saving role of A2M was first described in a bacterial protease induced septic shock animal models ^1-4. It was also shown that one of the major mechanisms for cause of septic shock followed by death was through the activation of plasma kallikrein-kinin system in which bradykinin was released from high molecular weight kininogen by bacterial protease^2-3. In that study it was clearly shown that bacterial protease can generate maximum amount of bradykinin within 5 seconds in plasma in the presence of kininase I and II inhibitor (ortho-phenthroloine) in vitro³. In presence of bradykinin receptor antagonist also showed complete inhibition of severe hypotension and shock followed by death in protease induced shock model³.

Proteins of the A2M family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates and invertebrates or even plant kingdom⁹. A shared suite of unique functional characteristics have been documented for the A2Ms of vertebrates, arthropods, and mollusks. The A2Ms of nematodes, arthropods, mollusks show significant sequence identity in key functional domains. Thus, the A2Ms comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution⁹. A2M prevents protease induced activation of coagulation by inhibiting thrombin and inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. Numerous growth factors, cytokines and hormones bind to A2M.  A2M also binds soluble beta-amyloid, of which it mediates degradation as seen in the brain of Alzheimer's disease (AD) patients. A2M is synthesized mainly in liver, but also locally by macrophages, fibroblasts, and adrenocortical cells.A2M is also produced in the brain where it binds multiple extracellular ligands and is internalized by neurons and astrocytes^23-28.

A2M: First line of defense protein in our innate immune system (suggestion)

Mechanism of inhibition of proteases by A2M (Table 2): Proteases are currently classified into six broad groups: Serine proteases, Threonine proteases, Cysteine proteases, Aspartate proteases, Metalloproteases and Glutamic acid proteases²⁹.

The protease inhibitors are of two classes³⁰, the active-site inhibitors and the α₂-macroglobulins. Inhibitors for the first class bind and inactivate the active site of the target protease. Proteins of the second class bind proteases by a unique molecular trap mechanism and deliver the bound protease to a receptor-mediated endocytic system for degradation in secondary lysosomes.

In human plasma, concentration of A2M is 2-4 mg/ml.  Its molecular size is 720 kDa. A2M is composed of four identical subunits bound together by -S-S- bonds. A2M has in its structure a 35 amino acid "bait" region. Proteases binding and cleaving the bait region become bound proteinase-A2M complex. The proteinase-A2M complex is recognized by macrophage receptors and cleared from the system²¹. 

CONCLUSION

In the description of innate immune system the role of A2M had never been described. Invasion of pathogen inside the body is the beginning of many disease processes. Microorganisms secrets proteases for invasion and A2M prevents  the protease activity in the physiological condition. Therefore, A2M could be considered as the first major component for defense in the innate immune system.

The status of A2M concentration in the body may be a very important predictor for the severity of many diseases. Normally many toxic products, in addition to proteases also binds with A2M and removed from the body.  Deficiency of A2M from its physiological concentration is not only risk for protease clearance, but also for clearance of other toxic products during disease conditions. Therefore, it is very important to measure plasma A2M and proteases in many diseases to know the status and progress of the disease. A2M could be a breakthrough life saving medicine to cure many diseases.

REFERENCES

1.    Khan MM and Scharko AM. The pathophysiology and treatment of septic shock: The a-2-macroglobulin hypothesis. 2012. (Online Journal: Rarediseaseindia.org): www.rarediseasesindia.org/septicshock

2.    Khan MM, Yamamoto T, Araki H, Ijiri H, Shibuya Y, Okamoto M., Kambara T. Pseudomonal elastase injection causes low vascular resistant shock in guinea pigs. Biochim Biophys Acta. 1993;1182:83-93.

3.    Khan MM, Yamamoto T, Araki H, Shibuya Y, Kambara T. Role of Hageman factor/kallikrein-kinin system in pseudomonal elastase-induced shock model.  Biochim. Biophys. Acta. 1993;1157:119-126.

4.    Khan MM, Shibuya Y, Nakagaki T, Kambara T, Yamamoto T. Alpha-2-macroglobulin as the major defense in acute pseudomonal septic shock model in guinea pigs.  Intl. J. Exp. Pathol. 1994;75:285-293.

5.    Khan MM, Shibuya Y, Kambara T, Yamamoto T.  Role of alpha-2-macroglobulin and bacterial elastase in guinea pig pseudomonal septic shock.  Intl. J. Exp. Pathol. 1995;76:21-28.

6.    Wang S, Wei X, Zhou J, Zhang J, Li K, Chen Q, Terek R, Fleming BC, Goldring MB, Ehrlich MG, Zhang G, Wei L. Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis. Arthritis Rheumatol. 2014 Jul;66(7):1843-1853.

7.    Khan MM. চিকিৎসাবিজ্ঞানে নতুন এক দিগন্ত উন্মোচন-(Focus) “A new horizon in Medical Science”- Published in Daily Inquilab, Nov 11. 2014 (language-bangali, newspaper in Bangladesh)

8.    Khan MM. জীবনরহ্মাকারী একটি প্রোটিন. “A life saving protein”. Published in in Weekly Thikana, Nov. 8, Page 8, 2014 (language-bangali, Newspaper in USA).

9.    Armstrong PB. Proteases and protease inhibitors: a balance of activities in host-pathogen interaction. Immunobiology. 2006; 211(4):263-281.

10.  Kong F, Singh RP. "Disintegration of Solid Foods in Human Stomach". 2008. Journal of Food Science 73 (5): R67–R80.

11.  Puente XS, Sanchez LM, Overall CV, Lopez-Otin C. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet 2003; 4 (7): 544-558.

12.  Seife C. Blunting nature's Swiss army knife. Science. 1997 Sep 12; 277(5332):1602-1603.

13.  Watanabe M, Hirano A, Stenglein S, Nelson J, Thomas G, Wong TC. Engineered serine protease inhibitor prevents furin-catalyzed activation of the fusion glycoprotein and production of infectious measles virus. J Virol. 1995 May;69(5):3206-10.

14.  Edinger TO, Pohl MO, Yángüez E, Stertz S. Cathepsin W Is Required for Escape of Influenza A Virus from Late Endosomes. MBio. 2015 Jun 9;6(3).

15.  Almeida-Paes R, de Oliveira LC, Oliveira MM, Gutierrez-Galhardo MC, Nosanchuk JD, Zancopé-Oliveira RM. Phenotypic characteristics associated with virulence of clinical isolates from the Sporothrix complex. Biomed Res Int. 2015;2015:212308.

16.  da Silva Júnior WS, de Godoy-Matos AF, Kraemer-Aguiar LG. Dipeptidyl Peptidase 4: A New Link between Diabetes Mellitus and Atherosclerosis? Biomed Res Int. 2015;2015:816164.

17.  Chinello P, Cicalini S, Pichini S, Pacifici R, Tempestilli M, Cicini MP, Pucillo LP, Petrosillo N.

Sildenafil and bosentan plasma concentrations in a human immunodeficiency virus- infected patient with pulmonary arterial hypertension treated with ritonavir-boosted protease inhibitor. Infect Dis Rep. 2015 Mar 16;7(1):5822.

18.  Raut R, Beesetti H, Tyagi P, Khanna I, Jain SK, Jeankumar VU, Yogeeswari P, Sriram D, Swaminathan S. A small molecule inhibitor of dengue virus type 2 protease inhibits the replication of all four dengue virus serotypes in cell culture. Virol J. 2015 Feb 8;12:16.

19.  Gowrishankar S, Yuan P, Wu Y, Schrag M, Paradise S, Grutzendler J, De Camilli P, Ferguson SM.Massive accumulation of luminal protease-deficient axonal lysosomes at Alzheimer's diseaseamyloid plaques. Proc Natl Acad Sci U S A. 2015 Jun 29. pii: 201510329. [Epub ahead of print]

20.  Påhlman LI, Jögi A, Gram M, Mori M, Egesten A. Hypoxia down-regulates expression of secretory leukocyte protease inhibitor in bronchial epithelial cells via TGF-β1. BMC Pulm Med. 2015 Mar 7;15:19.

21.  Borth W. a2 – Macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 1992, 6:3345-3353.

22.  McMahon MJ, Bowen M, Mayer AD, Cooper EH: Relation of alpha-2-macroglobulin and other antiproteases to the clinical features of acute pancreatitis. Am J Surg 1984;147:164-170

23.  Nezu T, Hosomi N, Aoki S, Deguchi K, Masugata H, Ichihara N, Ohyama H, Ohtsuki T, Kohno M, Matsumoto M. Alpha2-macroglobulin as a promising biomarker for cerebral small vessel disease in acute ischemic stroke patients. J Neurol. 2013 Oct; 260(10):2642-9.

24.  Zhang H, Song L, Li C, Zhao J, Wang H, Gao Q, Xu W. Molecular cloning and characterization of a thioester-containing protein from Zhikong scallop Chlamys farreri. Mol Immunol. 2007 Jul; 44(14):3492-500.

25.  Bätz T, Förster D, Luschnig S. The transmembrane protein Macroglobulin complement-related is essential for septate junction formation and epithelial barrier function in Drosophila.

Development. 2014 Feb; 141(4):899-908.

26.  Borisova EA, Gorbushin AM.Molecular cloning of α-2-macroglobulin from hemocytes of common periwinkle Littorina littorea. Fish Shellfish Immunol. 2014 May 14; 39(2):136-137.

27.  Marino R, Kimura Y, De Santis R, Lambris JD, Pinto MR.  Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis. Immunogenetics. 2002 Mar; 53(12):1055-64.

28.  Hammond JA, Nakao M, Smith VJ. Cloning of a glycosylphosphatidylinositol-anchored alpha-2-macroglobulin cDNA from the ascidian, Ciona intestinalis, and its possible role in immunity. Mol Immunol. 2005, Apr; 42(6):683-94.

29.  Abstract Goll, Darrel E., Valery F. Thompson, Hongqi Li, Wei Wei, and Jinyang Cong. The 

Calpain System. Physiol Rev.  2003; 83: 731–801.

30.  "Evolutionary families of peptidase inhibitors". Biochem. J. 378(Pt 3): 705–716.

 Submitted to Rare Diseases India on July 14, 2015.