Dr Umakhanth Venkatraman Girija

Job: Senior Lecturer (VC2020) and Programme Leader for MSc Advanced Biomedical Science

Faculty: Health and Life Sciences

School/department: School of Allied Health Sciences

Address: De Montfort University, The Gateway, Leicester, LE1 9BH

T: +44(0) 116 2577717

E: umakhanth.venkatramangirija@dmu.ac.uk

W: http://dmu.ac.uk/hls

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Personal profile

Dr Umakhanth Venkatraman Girija is a lecturer in Immunology in the School of Allied health Sciences, De Montfort University. He completed his PhD in Biochemistry from University of Oxford.

During his doctoral research, Umakhanth investigated the protein-protein interactions leading to the lectin pathway of complement activation, an important component of innate immune system. His postdoctoral work with University of Leicester expanded his interest into the molecular mechanism of classical pathway of complement activation and also on the role of lectins in immunity.

In addition to academic research, Umakhanth has six years of industrial research experience from Biocon, India, where he developed company’s first GMO producing recombinant therapeutic which is in market. He was a team leader for various molecular biology projects which were done in collaboration with multinational pharma companies from Europe and USA.

Currently, Umakhanth's research focuses on how microbial pathogens evade host complement attack and also on the role of adiponectins which has important cardio protective function. He has research collaborations with Universities of Leicester, Brunel and Warwick.

Research group affiliations

Biomedical and Environmental Health Group
Infectious Disease Research Group

Publications and outputs

Heme binding to human CLOCK affects interactions with the E-box
Heme binding to human CLOCK affects interactions with the E-box Freeman, S.L.; Kwon, H.; Portolano, N.; Parkin, G.; Venkatraman Girija, U.; Basran, J.; Fielding, A.; Fairall, L.; Svistunenko, D.; Moody, P.; Schwabe, J.; Kyriacou, C.; Raven, E. The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Freeman, S., Kwon, H., Portolano, N., Parkin, G., Venkatraman Girija, U., Basran, J., Fielding, A., Fairall, L., Svistunenko, D., Moody, P., Schwabe, J., Kyriacou, C. and Raven, E. (2019) Heme binding to human CLOCK affects interactions with the E-box. Proceedings of the National Academy of Sciences, 201905216.
Structure of the C1r-C1s interaction of the C1 complex of complement activation
Structure of the C1r-C1s interaction of the C1 complex of complement activation Almitairi, J.O.M.; Venkatraman Girija, U.; Furze, Christopher M.; Simpson-Gray, X.; Badakshi, F.; Marshall, Jamie E.; Schwaeble, W. J.; Mitchell, D. A.; Moody, P. C. E.; Wallis, R. The multiprotein complex C1 initiates the classical pathway of complement activation on binding to antibody–antigen complexes, pathogen surfaces, apoptotic cells, and polyanionic structures. It is formed from the recognition subcomponent C1q and a tetramer of proteases C1r2C1s2 as a Ca2+-dependent complex. Here we have determined the structure of a complex between the CUB1-EGF-CUB2 fragments of C1r and C1s to reveal the C1r– C1s interaction that forms the core of C1. Both fragments are Lshaped and interlock to form a compact antiparallel heterodimer with a Ca2+ from each subcomponent at the interface. Contacts, involving all three domains of each protease, are more extensive than those of C1r or C1s homodimers, explaining why heterocomplexes form preferentially. The available structural and biophysical data support a model of C1r2C1s2 in which two C1r-C1s dimers are linked via the catalytic domains of C1r. They are incompatible with a recent model in which the N-terminal domains of C1r and C1s form a fixed tetramer. On binding to C1q, the proteases become more compact, with the C1r-C1s dimers at the center and the six collagenous stems of C1q arranged around the perimeter. Activation is likely driven by separation of the C1r-C1s dimer pairs when C1q binds to a surface. Considerable flexibility in C1s likely facilitates C1 complex formation, activation of C1s by C1r, and binding and activation of downstream substrates C4 and C4b-bound C2 to initiate the reaction cascade. The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. Almitairi, J., Venkatraman Girija, U., Furze, C., Simpson-Gray, X., Badakshi, F., Marshall, J., Schwaeble, W., Mitchell, D., Moody, P. and Wallis, R. (2018) Structure of the C1r-C1s interaction of the C1 complex of complement activation. Proceedings of the National Academy of Sciences, 115(4), pp.768-773.
Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome
Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome Furze, Christopher M.; Gingras, Alexandre R.; Yoshizaki, Takayuki; Ohtani, Katsuki; Marshall, Jamie E.; Wallis, A. K.; Schwaeble, W. J.; El-Mezgueldi, Mohammed; Mitchell, D. A.; Moody, P. C. E.; Wakamiya, Nobutaka; Wallis, R.; Venkatraman Girija, U. BACKGROUND: Collectin-K1 (CL-K1, or CL-11) is a multifunctional Ca(2+)-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly(204)Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals. RESULTS: In this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca(2+) binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca(2+) during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder. CONCLUSIONS: We have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca(2+) binding during biosynthesis. Venkatraman Girija, U. et al. (2015) Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome. BMC Biology, 13 (1), pp. 27
Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin
Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin Risteli, M.; Ruotsalainen, H.; Bergmann, U.; Venkatraman Girija, U.; Wallis, R.; Myllyla, R. Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers. Risteli, M., Ruotsalainen, H., Bergmann, U., Venkatraman Girija, U., Wallis, R. and Myllyla, R. (2014) Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin. PLoS ONE, 9 (11), e113498
Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation
Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation Venkatraman Girija, U.; Gingras, A. R.; Marshall, J. E.; Panchal, R.; Sheikh, M. A.; Gal, P.; Schwaeble, W. J.; Mitchell, D. A.; Moody, P. C.; Wallis, R. Complement component C1, the complex that initiates the classical pathway of complement activation, is a 790-kDa assembly formed from the target-recognition subcomponent C1q and the modular proteases C1r and C1s. The proteases are elongated tetramers that become more compact when they bind to the collagen-like domains of C1q. Here, we describe a series of structures that reveal how the subcomponents associate to form C1. A complex between C1s and a collagen-like peptide containing the C1r/C1s-binding motif of C1q shows that the collagen binds to a shallow groove via a critical lysine side chain that contacts Ca(2+)-coordinating residues. The data explain the Ca(2+)-dependent binding mechanism, which is conserved in C1r and also in mannan-binding lectin-associated serine proteases, the serine proteases of the lectin pathway activation complexes. In an accompanying structure, C1s forms a compact ring-shaped tetramer featuring a unique head-to-tail interaction at its center that replicates the likely arrangement of C1r/C1s polypeptides in the C1 complex. Additional structures reveal how C1s polypeptides are positioned to enable activation by C1r and interaction with the substrate C4 inside the cage-like assembly formed by the collagenous stems of C1q. Together with previously determined structures of C1r fragments, the results reported here provide a structural basis for understanding the early steps of complement activation via the classical pathway. Venkatraman Girija, U., Gingras, A.R., Marshall, J.E., Panchal, R., Sheikh, M.A. Gál, P., Schwaeble, W.J., Mitchell, D.A. Moody, P.C. and Wallis R. (2013) Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation. Proceedings of the National Acadamy of Science of the U S A. Aug 20; 110 (34), pp. 13916-13920
The lectin pathway of complement activation is a critical component of the innate immune response to pneumococcal infection
The lectin pathway of complement activation is a critical component of the innate immune response to pneumococcal infection Ali, Y. M.; Lynch, N. J.; Haleem, K. S.; Fujita, T.; Endo, Y.; Hansen, S.; Holmskov, U.; Takahashi, K.; Stahl, G. L.; Dudler, T.; Venkatraman Girija, U.; Wallis, R.; Kadioglu, A.; Stover, C. M.; Andrew, P. W.; Schwaeble, W. J. The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci. Ali, Y.M. et al. (2012) The lectin pathway of complement activation is a critical component of the innate immune response to pneumococcal infection. PLoS Pathogolgy. 8 (7), e1002793
Structural basis of mannan-binding lectin recognition by its associated serine protease MASP-1: implications for complement activation
Structural basis of mannan-binding lectin recognition by its associated serine protease MASP-1: implications for complement activation Gingras, A. R.; Venkatraman Girija, U.; Keeble, A. H.; Panchal, R.; Mitchell, D. A.; Moody, P. C. E.; Wallis, R. Complement activation contributes directly to health and disease. It neutralizes pathogens and stimulates immune processes. Defects lead to immunodeficiency and autoimmune diseases, whereas inappropriate activation causes self-damage. In the lectin and classical pathways, complement is triggered upon recognition of a pathogen by an activating complex. Here we present the first structure of such a complex in the form of the collagen-like domain of mannan-binding lectin (MBL) and the binding domain of its associated protease (MASP-1/-3). The collagen binds within a groove using a pivotal lysine side chain that interacts with Ca(2+)-coordinating residues, revealing the essential role of Ca(2+). This mode of binding is prototypic for all activating complexes of the lectin and classical pathways, and suggests a general mechanism for the global changes that drive activation. The structural insights reveal a new focus for inhibitors and we have validated this concept by targeting the binding pocket of the MASP. Gingras, A.R., Venkatraman Girija, U., Keeble, A.H., Panchal, R., Mitchell, D.A., Moody, P.C.E. and Wallis R. (2011) Structural basis of mannan-binding lectin recognition by its associated serine protease MASP-1: implications for complement activation. Structure, 19 (11), pp. 1635-1643
Carbohydrate recognition and complement activation by rat ficolin-B
Carbohydrate recognition and complement activation by rat ficolin-B Venkatraman Girija, U.; Mitchell, D. A.; Roscher, S.; Wallis, R. Ficolins are innate immune components that bind to PAMPs and structures on apoptotic cells. Humans produce two serum forms (L- and H-ficolin) and a leukocyte-associated form (M-ficolin), whereas rodents and most other mammals produce ficolins-A and -B, orthologues of L- and M-ficolin, respectively. All three human ficolins, together with mouse and rat ficolin-A, associate with mannan-binding lectin-associated serine proteases (MASPs) and activate the lectin pathway of complement on PAMPs. By contrast, mouse ficolin-B does not bind MASPs and cannot activate complement. Because of these striking differences together with the lack of functional information for other ficolin-B orthologues, we have characterized rat ficolin-B, and compared its physical and biochemical properties with its serum counterpart. The data show that both rat ficolins have archetypal structures consisting of oligomers of a trimeric subunit. Ficolin-B recognized mainly sialyated sugars, characteristic of exogenous and endogenous ligands, whereas ficolin-A had a surprisingly narrow specificity, binding strongly to only one of 320 structures tested: an N-acetylated trisaccharide. Surprisingly, rat ficolin-B activated MASP-2 comparable to ficolin-A. Mutagenesis data reveal that lack of activity in mouse ficolin-B is probably caused by a single amino acid change in the putative MASP-binding site that blocks the ficolin-MASP interaction. Venkatraman Girija, U., Mitchell, D.A., Roscher, S. and Wallis R. (2011) Carbohydrate recognition and complement activation by rat ficolin-B. European Journal of Immunology, 41 (1), pp. 214-223
Engineering novel complement activity into a pulmonary-surfactant protein
Engineering novel complement activity into a pulmonary-surfactant protein Venkatraman Girija, U.; Furze, C.; Toth, J.; Schwaeble, W. J.; Mitchell, D. A.; Keeble, A. H.; Wallis, R. Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin.MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition. Venkatraman Girija, U., Furze, C., Toth, J., Schwaeble, W.J., Mitchell, D.A., Keeble, A.H. and Wallis, R. (2010) Engineering novel complement activity into a pulmonary-surfactant protein. The Journal of Biological Chemistry, 285 (14), pp. 10546-10552
Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement
Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement Phillips, A. E.; Toth, J.; Dodds, A. W.; Venkatraman Girija, U.; Furze, Christopher M.; Pala, E.; Sim, R.B.; Reid, K. B. M.; Schwaeble, W. J.; Schmid, R.; Keeble, A. H.; Wallis, R. The classical and lectin pathways of complement activation neutralize pathogens and stimulate key immunological processes. Both pathways are initiated by collagen-containing, soluble pattern recognition molecules associated with specific serine proteases. In the classical pathway, C1q binds to Ab-Ag complexes or bacterial surfaces to activate C1r and C1s. In the lectin pathway, mannan-binding lectin and ficolins bind to carbohydrates on pathogens to activate mannan-binding lectin-associated serine protease 2. To characterize the interactions leading to classical pathway activation, we have analyzed binding between human C1q, C1r, and C1s, which associate to form C1, using full-length and truncated protease components. We show that C1r and C1s bind to C1q independently. The CUB1-epidermal growth factor fragments contribute most toward binding, but CUB2 of C1r, but not of C1s, is also important. Each C1rs tetramer presents a total of six binding sites, one for each of the collagenous domains of C1q. We also demonstrate that subcomponents of the lectin and classical pathways cross-interact. Thus, although the stoichiometries of complexes differ, interactions are analogous, with equivalent contacts between recognition and protease subcomponents. Importantly, these new data are contrary to existing models of C1 and enable us to propose a new model using mannan-binding lectin-mannan-binding lectin-associated serine protease interactions as a template. Phillips, A.E., Toth, J., Dodds, A.W., Venkatraman Girija, U., Furze, C.M., Pala, E., Sim, R.B., Reid, K.B.M., Schwaeble, W.J., Schmid, R., Keeble, A.H. and Wallis, R. (2009) Analogous Interactions in Initiating Complexes of the Classical and Lectin Pathways of Complement. The Journal of Immunololgy, 182 (12), pp. 7708-7717

Click here to view a full listing of Umakhanth Venkatraman Girija's publications and outputs.

Research interests/expertise

Innate Immune System and complement pathways
Microbial pathogens and immune evasion strategies
Applied Biotechnology (recombinant protein expression)
Microbial strain engineering

Main techniques used in the laboratory include gene cloning, site-directed mutagenesis, recombinant protein expression in various host systems, protein purification, protein-protein interactions, assay development, cell culture and molecular microbiology (e.g. gene knockouts).

Areas of teaching

Immunology
Microbiology
Molecular Biology
Applied Biotechnology

Qualifications

PhD Biochemistry (University of Oxford)
MSc Microbiology (India)
PGCertHE (DMU)

Courses taught

BSc (Hons) Biomedical Science
BSc Medical Science
MSc Advanced Biomedical Science

Honours and awards

Best Poster Award, Complement UK meeting, New Castle, UK (October 2013)
Overseas Research Scholarship award for Doctoral study at University of Oxford (2006-08)
Biocontribute award in the industry for developing company’s first GMO and for successful collaborative research with pharma clients (December 2002 and December 2003)
Gold Medal for securing University First Rank in graduate studies in Microbiology (June1997 and June 1999)

Membership of external committees

Review Editor, the Editorial Board of Molecular Innate Immunity, a specialty of Frontiers in Immunology (since Nov 2015).

Conference attendance

4th Complement UK meeting, Leicester, UK, November 2016
11th International Conference on Innate Immunity, Olympia, Greece, June 2014
Complement UK meeting, New Castle, UK, October 2013
XII International Complement Workshop, Basel, Switzerland, October 2008
International Complement Meeting, MRC Immunochemistry Unit, Oxford, UK; July 2008
XI European meeting on Complement in Human Disease, Cardiff, UK; September, 2007
VI International Workshop on C1 and the Collectins, Seeheim, Germany; June 2006

Current research students

Miss Nisha Valand (PhD student, 1^st supervisor; Topic: Molecular immune evasion strategies of Candida tropicalis; Oct 2017 -)
Miss Emily Brunt (MSc research, Molecular Immunology, 1^st supervisor; Oct 2017 -)
Mr Medhanie Habtom (MSc research, Molecular Immunology, 1^st supervisor; Oct 2017 - )

Internally funded research project information

Project title: Schistosomiasis: Molecular investigation towards novel drug target and vaccine design

Value: £6959

Funding source: RIF7 (Research Investment Fund)

Start date: August 2015

End date: July 2016

Role in project: Principal Investigator

Project title: Protein Production Facility

Value: £46942

Funding source: RCIF2 (Research Capital Investment Funding 2)

Start date: December 2015

End date: July 2016

Role in project: Principal Investigator

Project title: Molecular characterization and identification of emerging parasitic pathogens in the environment in the midlands UK: Public health implications

Value: £21768

Funding source: RCIF2 (Research Capital Investment Funding 2)

Start date: December 2015

End date: July 2016

Role in project: Co-applicant (PI: Dr A Peña Fernández)

Project title: Coulter Counter and Cytospin

Value: £22494

Funding source: Capital bid

Start date: July 2016

Role in project: Co-applicant (PI: Dr Neenu Singh)