Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae)

International Journal of Biological Macromolecules 49 (2011) 85–92

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Biochemical and biophysical characterization of collagens of marine sponge, Ircinia fusca (Porifera: Demospongiae: Irciniidae) Ramjee Pallela a,c , Sreedhar Bojja b , Venkateswara Rao Janapala a,∗ a b c

Toxicology Unit, Biology Division, Indian Institute of Chemical Technology, Hyderabad 500 607, India Inorganic and Physical Chemistry Division, Indian Institute of Chemical Technology, Hyderabad 500 607, India Marine Bioprocess Research Center, Pukyong National University, Busan 608737, Republic of Korea

a r t i c l e

i n f o

Article history: Received 20 January 2011 Received in revised form 30 March 2011 Accepted 31 March 2011 Available online 9 April 2011 Keywords: Macromolecules Marine collagen Ircinia fusca Atomic Force Microscopy Gulf of Mannar

a b s t r a c t Collagens were isolated and partially characterized from the marine demosponge, Ircinia fusca from Gulf of Mannar (GoM), India, with an aim to develop potentially applicable collagens from unused and underused resources. The yield of insoluble, salt soluble and acid soluble forms of collagens was 31.71 ± 1.59, 20.69 ± 1.03, and 17.38 ± 0.87 mg/g dry weight, respectively. Trichrome staining, Scanning & Transmission Electron microscopic (SEM & TEM) studies confirmed the presence of collagen in the isolated, terminally globular irciniid filaments. The partially purified (gel filtration chromatography), non-fibrillar collagens appeared as basement type collagenous sheets under light microscopy whereas the purified fibrillar collagens appeared as fibrils with a repeated band periodicity of 67 nm under Atomic Force Microscope (AFM). The non-fibrillar and fibrillar collagens were seen to have affinity for anti-collagen type IV and type I antibodies raised against human collagens, respectively. The macromolecules, i.e., total protein, carbohydrate and lipid contents within the tissues were also quantified. The present information on the three characteristic irciniid collagens (filamentous, fibrillar and non-fibrillar) could assist the future attempts to unravel the therapeutically important, safer collagens from marine sponges for their use in pharmaceutical and cosmeceutical industries. © 2011 Elsevier B.V. All rights reserved.

1. Introduction During the last few years, there has been an intensive search for new pharmacologically active compounds and as a consequence, an interest in the biochemical, biophysical and molecular aspects of marine organisms in general and of sponges in particular, has also increased [1]. Gulf of Mannar (GoM) has been a storehouse of several biologically important compounds and many of them have been found to be useful antimicrobial, larvicidal, anticancerous and even antiretroviral (e.g., anti-HIV) agents, etc. [2–6]. Among the marine organisms of GoM, much diversity has been seen in the sponge species of the phylum Porifera and novel molecules derived from them. Sponges, a mass of cells formed of a porous skeleton made of organic (collagen fibres and/or spongin, especially in the

Abbreviations: GoM, Gulf of Mannar; AFM, Atomic Force Microscopy; SEM, Scanning Electron Microscopy; TEM, Transmission Electron Microscopy; BSE, Bovine Spongiform Encephalopathy; TSE, Transmissible Spongiform Encephalopathy; Hyp, Hydroxyproline; SS, Salt Soluble; AS, Acid Soluble; InS, Insoluble. ∗ Corresponding author. Tel.: +91 40 2719 3191/2720 5440; fax: +91 40 2719 3227. E-mail address: [email protected] (V.R. Janapala). 0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2011.03.019

case of the class Demospongiae) and inorganic (spicules) components, are the most primitive of multicellular animals (Metazoa) [6]. Most of the marine sponges produce a variety of secondary metabolites to prevent predation and fouling [7]. Apart from the secondary metabolites, tough bundles of collagen called natural collagen fibres have also been isolated from different marine sponges, e.g., various Ircinia species, Chondrosia reniformis and Suberites domuncula with great applications in pharmaceutical technology, cosmetics and nutrition and a high potency in tissue regeneration, especially after injuries [8]. Although the importance of safer marine originated collagen and its applications are increasing day by day, very little research has been done on biomaterial availability from marine sponge. Despite of the fact that ample of studies have reported about collagen in marine vertebrates and invertebrates, the main sources of industrial collagen are limited to those from bovine and pig skins [9,10]. However, the yield and quality of bovine collagen varies from batch to batch, which may elicit antigenic responses [11]. Owing to these challenges, reconsideration of industrial use of bovine collagen is mandatory to avoid the risks of BSE (bovine spongiform encephalopathy) and TSE (transmissible spongiform encephalopathy) [8]. As an alternative, sponge collagens may prove to be a better and safer biomaterial but till date

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Fig. 1. Map showing the sampling location (Mandapam and Palk Bay) of Ircinia fusca near Gulf of Mannar, India.

only few investigations have yielded information on the ultrastructure and biochemical properties of sponge collagens [12]. The present research aimed to isolate and characterize the irciniid collagens for the first time from the marine sponge, Ircinia fusca of Gulf of Mannar, India. The surface of these collagen filaments were analyzed by Scanning Electron Microscopy and the characteristic repeated band periodicity was elucidated by Transmission Electron Microscopy. Chromatographically purified fibrillar and non-fibrillar collagens were subjected to Atomic Force and Light Microscopy, respectively to investigate the ultrastructure and morphological organization, and these collagens were immunologically characterized by western blotting. Our results present the ultrastructure and organization of irciniid collagens and provide an advanced model for exploring sponge collagens of Indian marine origin. These studies can further extend to other biologically important sponges that are capable to produce chemical as well as protein therapeutic molecules. 2. Materials and methods 2.1. Collection of sponges Sponges were carefully collected from the waters of Mandapam region, Gulf of Mannar [between 78◦ 05 to 79◦ 30 E Longitude and 8◦ 47 to 9◦ 15 N Latitude], India (Fig. 1). The sponges were gently removed from the substratum and placed in plastic bags underwater, then transferred into large containers of aerated seawater for transport to the laboratory. Before analysis, the sponges were thoroughly cleaned for mechanical removal of foreign materials followed by repeated washing with artificial sea water. The voucher specimens were submitted to National Institute of Oceanography (NIO), Goa for depository purpose, and were identified as I. fusca Carter 1880 (Class: Demospongiae; order: Dictyoceratida, Family: Irciniidae) at Vizhinjam Research Centre of Central Marine Fisheries Research Institute (ICAR),Vizhinjam, Thiruvananthapuram, India. 2.2. Macromolecular estimations of I. fusca Fresh tissue of Ircinia was separately processed for estimating macromolecular content viz., carbohydrates, proteins and lipids

in individual samples. After prior washing of the sponge tissue with 0.1 M PBS (pH 7.2), macromolecular composition was analyzed using the following standard procedures. Protein was estimated according to the method of Bradford [13] with bovine serum albumin as standard, total carbohydrate content using the phenol–sulfuric acid method of Taylor [14], whereas the total lipid content was measured by using a 1:2 mixture of chloroform:methanol according to the method of Brooks et al. [15]. 2.3. Histological studies of I. fusca Masson’s Trichrome procedure involves three important stains, i.e., Weigert’s hematoxylin, Biebrich scarlet-acid fuchsin and aniline blue. This staining is used to differentiate collagen with other tissue components. Initially, samples were fixed in 10% buffered formalin stored at 4 ◦ C for 24–72 h. After fixation, tissues were embedded in paraffin and subsequently sectioned to a thickness of 5–10 ␮m using a rotary microtome. Tissue sections were then deparaffinized and rehydrated in graded ethanol solutions (100–70%), and subjected to a modified Masson’s Trichrome staining procedure [16]. The stained glass slides were observed carefully under Reichert-Jung light microscope to observe the blue color acquired by collagen. 2.4. Extraction and quantification of collagens from I. fusca Different forms of collagens viz., salt soluble (SS), acid soluble (AS) and insoluble (InS) collagens from I. fusca were extracted by the modified method of Prockop [17]. In addition, modified method of Siddiqi and Alhomida [18] was used to quantify total tissue collagen via the estimation of hydroxyproline (Hyp), a marker iminoacid (about 13%) in collagen. Total collagen and different forms (SS, AS and InS) of collagen were quantified according to the standardized calculation by Neuman and Logan [19]. The following formula was used to calculate the collagen content. collagen content (mg of collagen/g dry weight of sponge tissue) = 7.46 ×



mg of hydroxyproline g dry weight of sponge tissue



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Fig. 2. Under water digital image of Ircinia fusca attached to the substratum (scale bar: 50 cm).

where the factor 7.46 = ratio of the weight of collagen and weight of hydroxyproline. 2.5. Isolation of collagens from I. fusca Collagens were isolated according to the method of Swatschek et al. [8] with slight modifications of sonicating the sponge tissue mass before the homogenization procedure. This facilitated the release of intertwined insoluble collagen like filaments (often referred to as irciniid filaments) from the sponge matrix, which were further subjected to SEM and TEM analyses. In addition, 6 M urea (instead of 8 M) has been used to retain non-filamentous collagens along with the soluble and insoluble collagens in the subsequent extraction and purification procedures. 2.6. Purification of collagens A modified procedure of Diehl-Seifert et al. [20] was followed for the purification of functionally active collagens from I. fusca by gel filtration on a Sepharose 4B column (5 cm × 30 cm) using the 100 mM Tris–HCl buffer (pH 9.0) as eluant. Collagens of fibrillar and non-fibrillar nature were freed in the post column extractions, which were then characterized by microscopic and immunoblotting analyses. 2.7. Characterization of collagens from I. fusca 2.7.1. Irciniid filament studies 2.7.1.1. Scanning Electron Microscopy. Scanning Electronic Microscopy was performed based on the principles of Bozzola and Russell [21]. Tissue sections and collagen like filaments of I. fusca were mounted over the stubs and fixed with 4% aqueous Osmium tetroxide vapors for 2 h. A thin layer of Platinum (palladium) was coated over the samples using an automated sputter coater (JEOL JFC-1600) for 4 min approximately. Then the samples were scanned under Scanning Electron Microscope (Model: JOEL JSM-5600) at various magnifications. 2.7.1.2. Transmission Electron Microscopy. The isolated pellet of irciniid fibres were fixed in 2.5% gluteraldehyde in 0.05 M phosphate buffer (pH 7.2) for 24 h at 4 ◦ C and re-fixed by 2% Osmium

tetroxide in the same buffer for 2 h. Samples were dehydrated in a series of graded alcohol, infiltrated and then embedded in Spurr’s resin [22]. Samples were subsequently subjected to ultra thin sectioning with a glass knife fitted on an ultra Microtome (LEICA ULTRA CUT: UCT-GA-D/E-1/00). Ultra thin sections of 50–70 nm thickness were mounted on grids and the sections were stained with saturated aqueous uranyl acetate and counter stained with 4% lead citrate. The mounted grids were scanned for any collagens at various magnifications under HITACHI (H-7500) Transmission Electron Microscope.

2.7.2. Non-fibrillar collagens 2.7.2.1. Light microscopy. The purified collagens from the Sepharose 4B column were observed directly under high resolution light microscope (POLYVAR, Reichert-Jung) for any basement type sheets of collagens to generate a preliminary understanding of the general morphological appearance of purified sponge collagens.

2.7.3. Fibrillar collagens 2.7.3.1. Atomic Force Microscopy. The purified sample of fibrils from I. fusca were diluted suitably to disperse the individual fibrils on to the smoother surface of the mica block adhered to the circular glass disc. After drying of the sample, the glass disc was scanned under Atomic Force Microscope (Model: VEECO, NANOSCOPE IV) for analyzing the band periodicity of the fibrils.

2.7.4. Electrophoresis (SDS-PAGE) and Western blotting of purified collagens Post column collagen extractions from I. fusca were analyzed by SDS-PAGE under reducing conditions according to the method of Laemmli [23]. Electrophoreses were performed in a Mini-Protean electrophoresis unit (Bio-Rad Laboratories) using a 12% acrylamide separating gel and a 4% acrylamide stacking gel. The proteins were transferred from the gel to a PVDF membrane (Millipore) and after the trans-blotting, subsequent western blotting steps were performed according to the method of Towbin et al. [24]. Immunocompatibility of the sponge collagens to the monoclonal anti mouse type IV and type I collagen antibodies (Sigma) raised against human type IV and I collagens, respectively, was subsequently determined.

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Fig. 3. Light micrographs of (a) Trichrome stained collagen like filaments, and (b) tissue Cryo-section of Ircinia fusca. Blue colored areas in both the samples (a and b) indicated the presence of collagen. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

3. Results

3.4. Histological studies of I. fusca

3.1. Gross morphological study of I. fusca

As in the case of Spongia graminea, ramifying reticular fibrous network formed of free elongated collagen like filaments was typically observed in I. fusca. Bulky amorphous debris was scattered throughout the mesohyl indicating a fibre skeleton of moderate density. Though the substantial presence of stones, sand particles and high spicular content of the Ircinia tissue renders the sectioning by microtome a bit cumbersome, the irciniid filaments as well as the tissue mass got stained adequately and appeared in blue color under high resolution light microscope (POLYVAR, Reichert-Jung) (Fig. 3).

I. fusca often remains attached to a stony substratum in the form of huge mounds or boulders, with a conulose surface and appears grayish brown to dark grey or even to black in the polluted areas (Fig. 2). This species is available even at the inshore regions, but with a diverse external appearance due to the habitat disturbances by regular local trawlers of GoM. Sponge body is elastic and leathery in consistency and very difficult to tear because of the impregnated and intertwined irciniid filaments, which is the characteristic identification feature of this species.

3.5. Filamentous collagens (Irciniid filaments) 3.2. Macromolecular estimations of I. fusca According to the results obtained (Table 1) I. fusca contains protein (139.46 ± 6.97 mg/g dry sponge wt) as highest of all the macromolecules, followed by lipids (11.29 ± 0.57 mg/g dry sponge wt) and lastly by carbohydrates (pentose and hexose as, 3.96 ± 0.20 and 2.63 ± 0.13 mg/g dry sponge wt, respectively).

3.3. Hydroxyproline and collagen content After prior estimation of hydroxyproline based on the standard graph values, total collagen of I. fusca was calculated as 123.68 ± 6.18 mg/g dry sponge wt. In addition, the differential extractions and consequent quantification of the Irciniid collagens evidenced the presence of higher insoluble form of collagen (31.71 ± 1.59 mg/g dry sponge wt), than other two forms of collagens [salt soluble (SS) and acid soluble (AS) as 20.69 ± 1.03 and 17.38 ± 0.87 mg/g dry sponge wt, respectively], which would be due to the possession of higher collagenous, irciniid filaments by I. fusca.

3.5.1. Scanning Electron Microscopy Scanning Electron Microscopy of the ectosomal portion of intact sponge tissue revealed the skeletal make up and orientation of fibrillar material and the individual collagen like fibres embedded in extra cellular matrix of I. fusca, which were very diverse in their form. SEM pictures revealed that I. fusca possess huge amounts of spicules, sand particles and other debris entangling the mesh formed with the interlocking of the irciniid filaments (Fig. 4). After prior agitation and passing the ultrasonic waves through the tissue pieces, Ircinia tissue appeared to be packed with bundle of fibres ranging from 1.72 to 3.49 ␮m in diameter. The non-irciniid tissue clearly denotes the presence of enormous elongated fibres interlocked with their terminal knobs that resembles the terminal globular domains of the collagen fibrils in their form. The terminal globular domains of ∼10 ␮m diameter could form the interlocking grip to strengthen the tough skeleton of Ircinia. 3.5.2. Transmission Electron Microscopy The filaments of I. fusca were subjected to ultra thin sectioning and subsequent TEM (Fig. 5). It is evident from the results that these filaments were composed of parallel striated collagen fibrils with

Table 1 Macromolecular composition of sponge, Ircinia fusca. Carbohydrates Hexose

2.63 ± 0.13 a b

Lipids Pentose

3.96 ± 0.2

Collagena

Protein

Different forms of collagen

11.29 ± 0.56

Total collagen

SSb

ASb

InSb

20.69 ± 1.03

17.38 ± 0.87

31.71 ± 1.59

Data presented as mean ± SE of mg/g freeze-dried sponge weight. SS, AS and InS are salt soluble, acid soluble and insoluble forms of collagens, respectively.

123.68 ± 6.18

139.46 ± 6.97

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Fig. 4. Scanning electron micrographs of Ircinia fusca. (a) Section of intact sponge tissue; (b) agitated tissue without spicules/other debris showing the compacted filaments; (c) free elongated and knobbed filaments and (d) magnified images of the filaments, showing annular grooves formed by the tightly packed fibrils and the nodular, wavy surface of the terminal knob.

repeated band periodicity, a characteristic feature of collagens. At higher magnifications, the protofibrillar bands were found to be cross-linked by the proteoglycan connections that support the fibrillar rigidity (Fig. 5a). In addition, the section of the terminal knob of the filament exhibited granular appearance of collagen fibrils with a wavy/undular cuticle covering the fibrils (Fig. 5b). 3.6. Non-fibrillar collagens 3.6.1. Light Microscopy Transparent sheets of collagenous material from the purified fractions of the irciniid collagens were observed under light microscopy, which resembles the basement type non-fibrillar collagens of vertebrate origin (Fig. 6).

3.7. Fibrillar collagens 3.7.1. Atomic Force Microscopy In this paper, we have attempted to provide an insight to reveal the form and nature of the marine sponge collagens by application of the Atomic Force Microscopy (AFM). It is evidenced by AFM that collagen fibrils of I. fusca possessed a repeated band periodicity of 67 nm (Fig. 7). 3.8. Electrophoresis (SDS-PAGE) and Western blotting of purified collagens The purified collagens from I. fusca, when electrophoresed, showed specific bands (Fig. 8), one above 97 kDa and the other

Fig. 5. Transmission electron micrographs of isolated Irciniid filaments of Ircinia fusca. (a) Longitudinal section of the filament showing the isometric fibrillar repetition of band periodicity, as observed in typical collagen fibril and (b) cross section of the filament showing granular layered appearance of micro fibrillar structures covered by outer cuticular membrane.

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Fig. 6. Light micrographs of the purified non-fibrillar, basement type, collagenous sheets of Ircinia fusca. (a) Low magnification image of collagenous sheets and (b) high magnification of sheets of compact striations.

Fig. 7. Atomic force micrographs of the purified fibrillar collagens from Ircinia fusca. (a) Deflection mode height image of a collagen fibril with a typical band spacing of 67 nm [scan size 1 ␮m × 1 ␮m]; (b) amplitude signal showing the distance between the protrusions of two bands; (c) low scan phase image of the fibrils showing the repeated band periodicity [scan size 4 ␮m × 4 ␮m] and (d) amplitude image of collagen fibrils with repeated bands [scan size 0.5 ␮m × 0.5 ␮m].

Fig. 8. Western blot analyses of the purified fibrillar and non-fibrillar collagens from Ircinia fusca, showing specificity with monoclonal anti Type I (a) and Type IV (b) collagen antibodies, respectively. Lane 1, protein marker; lane 2, collagen band on PAGE gel; and lane 3, electro-blotted collagen on PVDF membrane.

in between 170 and 212 kDa marks as compared to the protein markers. After transblotting and consequent WB treatments, it was found that these bands had specificity for anti collagen type IV and type I antibodies, respectively. Though the type IV collagen is a non-fibrillar and network forming collagen, the presence of this variety of basement type collagens in the present study was also proven along with the fibrillar (Type I) collagens.

4. Discussion The genus Ircinia contains around 100 species with an excess of 40 published species worldwide, although the integrity is uncertain often because of poor diagnoses. Morphologically, I. fusca is of infundibuliformis type, inconspicuous species characterized by its simple, encrusting form [25]. Typically, I. fusca has a crustose to irregular massive body form with raised oscula whose surface

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is conulose (with conules 2–3 mm in height and 3–4 mm apart) and unarmored bearing fistules. This species would be difficult to identify in the field and could easily be confused with other Dictyoceratid species. However, the body is not upright and digitate like that of other Indopacific irciniid species. As its hard skeleton is non-absorbent due to the presence of thick elongated and terminally knobbed filaments, untrained divers can easily confuse it with a ‘good sponge’. Although this species shares similar features with other irciniids, fibres of I. fusca are well developed and appear in light brownish color, opaque and hollow. Characteristically, I. fusca possesses the irciniid filaments/fibres with a diverse range of diameter and moderate lepidocrocite deposition on the filaments when compared to the other species like I. fasciculata. Genomic and complementary DNA studies have shown that proteinaceous fibrous materials (collagen and spongin) contain the classic collagenous Gly-Xaa-Yaa motif, where Hydroxyproline (Hyp) occupies any one of the positions in the triplet motif, other than that of Glycine (Gly) [26]. It is well known that collagen is the only intercellular organic framework and amounts to approximately 10% of the total organic matter in Demospongiae [27], which was experimentally proven for the collagen content of I. fusca (≈7%) in the present study. It is also evidenced previously that not all the organic components were resolved for sponge biochemical content which can be correlated to the growth rate potential [28]. Our study determined the macromolecular content of I. fusca which resolved the significant correspondence with collagen content of the sponge species. Spicule formation is generally accomplished by specialized cells called sclerocytes that supply mineral ions or particles associated with organic macromolecules like protein, carbohydrates, lipids and by the nucleic acids to a limited extent [29]. Aizenberg et al. [30] concluded that intra-crystalline macromolecules play an important role in modulation of the morphologies of forming biogenic crystals that align to form canalicular tracts of the sponge skeleton and this property is defined directly or indirectly by apposition and deposition of macromolecules. Collagen arrangements and rearrangements play a major role in the adhesion and tissue growth in animals [31]. Although the presence of sponge collagen has been histologically proved, the characteristic insolubility and mineralization causes methodological problems while isolating and studying the collagen from the collected sponges [32,33]. Despite of the aforementioned impediments, marine demosponges were chosen for the present study because of their potentiality of producing novel collagens towards various medicinal and cosmeceutical applications [8]. The existence of collagen in marine as well as freshwater sponges was first proved electron microscopically in 1980s [20,34]. Investigations regarding the fine structure and physicochemical properties of collagen of the marine sponge C. reniformis Nardo started with the studies by Garrone et al. [35]. As defined earlier by the illustrations of Heinemann et al., the holography of TEM, SEM and AFM has provided substantial information on 3D ultrastructure and organization of collagens from marine sponges [12,36]. Our microscopic studies have also revealed some fascinating facts about the collagen content of I. fusca, as stated in the results section. In general, as both the structure and fibrillogenesis of fibrillar collagens remain poorly characterized, AFM has rapidly become a widely used technique for characterizing surface properties of these biologically important macromolecules [37]. The fibril forming types of collagen (type I and III) are abundant in most extracellular matrices whereas type IV collagen, a non-fibrillar network-forming collagen is a major component of basement membrane [38]. From the first multicellular animals (the glass sponges) to humans, only two collagen types appear to have been conserved, i.e., the fibrillar and the BM collagens [39]. From our results, it is certain that the non-fibrillar collagens are also present in I. fusca, as some portion of this type of collagen (Type IV)

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has shown immunocompatibility in western blotting. The present data is correlated with the earlier reports which suggest the existence of different type of collagen molecules within the species as well as tissues in the form of homotypic, heterotypic or even mixed. The occurrence of these collagens in sponge tissue has been reported to be heterotypic and it is believed that one of the polypeptide chains have been recognized in western blot justifying slight differences in the molecular weights as observed in the present study and previous studies of Bairati and Gioria [40]. AFM images of a native collagen type I fibril is generally with a banding periodicity of 67 nm with a width of 300 nm and a height of 20 nm, as observed in the purified sample of I. fusca. The native collagen type I fibrils differ from the Fibrous Long Spacing (FLS) fibrils, which possess a periodicity >67 nm, and their dimension is usually around 150–300 nm [41]. The formation of the type I fibrils and thus the band periodicity, generally varies along the axis of the fibril but sometimes due to the mechanical and the physiological changes, the band periodicity may vary even within the fibril. The presence of type I collagen in marine demosponges further opens the gateway to unravel the application of this most abundant collagen. Moreover the presence of a non-fibrillar network forming collagen (type IV collagen) in our study indicates the significance of I. fusca derived collagen in basement type collagen applications. The presence of immunocompatible collagens in marine sponges could form a basis for the followers to understand the form and type of the collagens so that specific genes involved in the production of safer collagens can be targeted. 5. Conclusions In the present study, the collagens of the marine sponge I. fusca were differentially examined to obtain continuous information on its biochemical and biophysical aspects. After implying a proper purification procedure, we have performed Atomic Force Microscopy of the Indian marine sponge collagens for the first time. The evidence of the presence of fibrillar collagens obtained in this present study could have tremendous impact in the applied sciences. Moreover the information on the biochemical and immunological make up of I. fusca, as reported in this study, could ascertain the benefits of sponge collagen to mankind in the future course of the study. In addition, the development of in vitro sponge cell, fragment and whole sponge culture/cultivation ex situ and in situ presents a valuable key for targeting the potential pharmaceutical and cosmeceutical secondary metabolites and more importantly the collagens from marine sponges. Further studies on the sponge collagens in terms of their biosynthetic regulation and interaction with other proteins might bring better understanding of the dynamic regulation of the structure and function of type I and IV collagens in marine collagens in general and I. fusca in particular. Acknowledgments The authors are thankful to the Director, IICT for providing the facilities and constant encouragement throughout the study. This work was supported by Task-force project (CMM 004) of Council of Scientific and Industrial Research (CSIR), Government of India. The author, RP is thankful to CSIR, for the award of senior research fellowship. References [1] D. Sipkema, R. Osinga, W. Schatton, D. Mendola, J. Tramper, R. Wijffels, Biotechnol. Bioeng. 90 (2005) 201–222. [2] T.V. Goud, N.S. Reddy, N.R. Swamy, T.S. Ram, Y. Venkateswarlu, Biol. Pharm. Bull. 26 (2003) 1498–1501.

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