Tartrate-Resistant Acid Phosphatase 5b: a Serum Marker of Bone Resorption

Bone is a physiologically dynamic tissue being constantly regenerated throughout life as a consequence of bone turnover by bone-resorbing osteoclasts and bone-forming osteoblasts. In certain bone diseases, such as osteoporosis, the imbalance in bone turnover leads to bone loss and increased fracture risk. Measurement of bone mineral density (BMD) predicts the risk of fracture, but also biochemical markers of bone metabolism have been suggested to be suitable for prediction of fractures and monitoring the efficacy of antiresorptive treatment. Tartrate-resistant acid phosphatase 5b (TRACP 5b) is an enzyme released from osteoclasts into the circulation, from where it can be detected kinetically or immunologically. Conventional assays for serum total TRACP were spectrophotometric and suffered from interference by other acid phosphatases and non-osteoclastic TRACP 5a isoform. Our aim was to develop novel immunoassays for osteoclastic TRACP 5b. Serum TRACP 5b levels were elevated in individuals with high bone turnover, such as children, postmenopausal women, patients with osteoporosis, Paget’s disease and breast cancer patients with bone metastases. As expected, hormone replacement therapy (HRT) in postmenopausal women decreased the levels of serum TRACP 5b. Surprisingly, the highest TRACP 5b levels were observed in individuals with rare autosomal dominant osteopetrosis type II (ADO2), which is characterized by high BMD and fracture risk with simultaneously elevated levels of deficient osteoclasts. In ADO2 patients, elevated levels of serum TRACP 5b were associated with high fracture frequency. It is likely that serum TRACP 5b reflects the number of inactive osteoclasts in ADO2. Similar results supporting the hypothesis that TRACP 5b would reflect the number of osteoclasts instead of their activity were observed with cultured osteoclasts and in animal models. Novel TRACP 5b immunoassays may prove to be of value either as independent or combinatory tools with other bone metabolic markers and BMD measurements in clinical practice and bone research.

Development of 3D super-resolution tomographic STED microscopy and its application to studies on bone resorption

In this doctoral thesis, a tomographic STED microscopy technique for 3D super-resolution imaging was developed and utilized to observebone remodeling processes. To improve upon existing methods, wehave used a tomographic approach using a commercially available stimulated emission depletion (STED) microscope. A certain region of interest (ROI) was observed at two oblique angles: one at a standard inverted configuration from below (bottom view) and another from the side (side view) via a micro-mirror positioned close to the ROI. The two viewing angles were reconstructed into a final tomogram. The technique, named as tomographic STED microscopy, was able to achieve an axial resolution of approximately 70 nm on microtubule structures in a fixed biological specimen. High resolution imaging of osteoclasts (OCs) that are actively resorbing bone was achieved by creating an optically transparent coating on a microscope coverglass that imitates a fractured bone surface. 2D super-resolution STED microscopy on the bone layer showed approximately 60 nm of lateral resolution on a resorption associated organelle allowing these structures to be imaged with super-resolution microscopy for the first time. The developed tomographic STED microscopy technique was further applied to study resorption mechanisms of OCs cultured on the bone coating. The technique revealed actin cytoskeleton with specific structures, comet-tails, some of which were facing upwards and some others were facing downwards. This, in our opinion, indicated that during bone resorption, an involvement of the actin cytoskeleton in vesicular exocytosis and endocytosis is present. The application of tomographic STED microscopy in bone biology demonstrated that 3D super-resolution techniques can provide new insights into biological 3D nano-structures that are beyond the diffraction-limit when the optical constraints of super-resolution imaging are carefully taken into account.

Osteoclastic tartrate-resistant acid phosphatase 5b. Diagnostic use and biological significance in bone physiology

The skeleton undergoes continuous turnover throughout life. In women, an increase in bone turnover is pronounced during childhood and puberty and after menopause. Bone turnover can be monitored by measuring biochemical markers of bone resorption and bone formation. Tartrate-resistant acid phosphatase (TRACP) is an enzyme secreted by osteoclasts, macrophages and dendritic cells. The secreted enzyme can be detected from the blood circulation by recently developed immunoassays. In blood circulation, the enzyme exists as two isoforms, TRACP 5a with an intact polypeptide chain and TRACP 5b in which the polypeptide chain consists of two subunits. The 5b form is predominantly secreted by osteoclasts and is thus associated with bone turnover. The secretion of TRACP 5b is not directly related to bone resorption; instead, the levels are shown to be proportional to the number of osteoclasts. Therefore, the combination of TRACP 5b and a marker reflecting bone degradation, such as C-terminal cross-linked telopeptides of type I collagen (CTX), enables a more profound analysis of the changes in bone turnover. In this study, recombinant TRACP 5a-like protein was proteolytically processed into TRACP 5b-like two subunit form. The 5b-like form was more active both as an acid phosphatase and in producing reactive oxygen species, suggesting a possible function for TRACP 5b in osteoclastic bone resorption. Even though both TRACP 5a and 5b were detected in osteoclasts, serum TRACP 5a levels demonstrated no change in response to alendronate treatment of postmenopausal women. However, TRACP 5b levels decreased substantially, demonstrating that alendronate decreases the number of osteoclasts. This was confirmed in human osteoclast cultures, showing that alendronate decreased the number of osteoclats by inducing osteoclast apoptosis, and TRACP 5b was not secreted as an active enzyme from the apoptotic osteoclasts. In peripubertal girls, the highest levels of TRACP 5b and other bone turnover markers were observed at the time of menarche, whereas at the same time the ratio of CTX to TRACP 5b was lowest, indicating the presence of a high number of osteoclasts with decreased resorptive activity. These results support the earlier findings that TRACP 5b is the predominant form of TRACP secreted by osteoclasts. The major source of circulating TRACP 5a remains to be established, but is most likely other cells of the macrophage-monocyte system. The results also suggest that bone turnover can be differentially affected by both osteoclast number and their resorptive activity, and provide further support for the possible clinical use of TRACP 5b as a marker of osteoclast number.

Intracellular membrane trafficking in Osteoclasts The role of direct Rab protein – Rac 1 interactions in the targeting of intracellular vesicles

Osteoclasts are cells responsible for bone resorption. These cells undergo extensive membrane re-organization during their polarization for bone resorption and form four distinct membrane domains, namely the ruffled border, the basolateral membrane, the sealing zone and the functional secretory domain. The endocytic/biosynthetic pathway and transcytotic route(s) are important for the resorption process, since the endocytic/biosynthetic pathway brings the specific vesicles to the ruffled border whereas the transcytotic flow is believed to transport the degraded bone matrix away from the resorption lacuna to the functional secretory domain. In the present study, we found a new transcytotic route from the functional secretory domain to the ruffled border, which may compensate membrane loss from the ruffled border during the resorption process. We also found that lipid rafts are essential for the ruffled border-targeted late endosomal pathways. A small GTP-binding protein, Rab7, has earlier been shown to regulate the late steps of the endocytic pathway. In bone-resorbing osteoclasts it is involved in the formation of the ruffled border, which displays several features of late endosomal membranes. Here we discovered a new Rab7-interacting protein, Rac1, which is another small GTP-binding protein and binds to the GTP-form of Rab7 in vitro. We demonstrated further that Rab7 colocalizes with Rac1 at the fusion zone of the ruffled border in bone-resorbing osteoclasts. In other cell types, such as fibroblast-like cells, this colocalization is mainly perinuclear. Because Rac1 is known to control the actin cytoskeleton through its effectors, we suggest that the Rab7-Rac1 interaction may mediate late endosomal transport between microtubules and microfilaments, thus enabling endosomal vesicles to switch tracks from microtubules to microfilaments before their fusion to the ruffled border. We then studied the role of Rab-Rac1 interaction in the slow recycling pathway. We revealed that Rac1 also binds directly to Rab11 and to some other but not all Rab-proteins, suggesting that Rab-Rac1 interaction could be a general regulatory mechanism to direct the intracellular vesicles from microtubule mediated transport to actin filament mediated transport and vice versa. On the basis of our results we thus propose a new hypothesis for these GTPases in the regulation of intracellular membrane flow.

Non-resorbable glass fibre-reinforced composite with porous surface as bone substitute material: Experimental studies in vitro and in vivo focused on bone-implant interface

The development of load-bearing osseous implant with desired mechanical and surface properties in order to promote incorporation with bone and to eliminate risk of bone resorption and implant failure is a very challenging task. Bone formation and resoption processes depend on the mechanical environment. Certain stress/strain conditions are required to promote new bone growth and to prevent bone mass loss. Conventional metallic implants with high stiffness carry most of the load and the surrounding bone becomes virtually unloaded and inactive. Fibre-reinforced composites offer an interesting alternative to metallic implants, because their mechanical properties can be tailored to be equal to those of bone, by the careful selection of matrix polymer, type of fibres, fibre volume fraction, orientation and length. Successful load transfer at bone-implant interface requires proper fixation between the bone and implant. One promising method to promote fixation is to prepare implants with porous surface. Bone ingrowth into porous surface structure stabilises the system and improves clinical success of the implant. The experimental part of this work was focused on polymethyl methacrylate (PMMA) -based composites with dense load-bearing core and porous surface. Three-dimensionally randomly orientated chopped glass fibres were used to reinforce the composite. A method to fabricate those composites was developed by a solvent treatment technique and some characterisations concerning the functionality of the surface structure were made in vitro and in vivo. Scanning electron microscope observations revealed that the pore size and interconnective porous architecture of the surface layer of the fibre-reinforced composite (FRC) could be optimal for bone ingrowth. Microhardness measurements showed that the solvent treatment did not have an effect on the mechanical properties of the load-bearing core. A push-out test, using dental stone as a bone model material, revealed that short glass fibre-reinforced porous surface layer is strong enough to carry load. Unreacted monomers can cause the chemical necrosis of the tissue, but the levels of leachable resisidual monomers were considerably lower than those found in chemically cured fibre-reinforced dentures and in modified acrylic bone cements. Animal experiments proved that surface porous FRC implant can enhance fixation between bone and FRC. New bone ingrowth into the pores was detected and strong interlocking between bone and the implant was achieved.

Acid-base Balance and Oxidative Metabolism in Calcified Tissues

The calcified tissues, comprising bone and cartilage, are metabolically active tissues that bind and release calcium, bicarbonate and other substances according to systemic needs. Understanding the regulation of cellular metabolism in bone and cartilage is an important issue, since a link between the metabolism and diseases of these tissues is clear. An essential element in the function of bone-resorbing osteoclasts, namely regulation of bicarbonate transport, has not yet been thoroughly studied. Another example of an important but at the same time fairly unexplored subject of interest in this field is cartilage degeneration, an important determinant for development of osteoarthritis. The link between this and oxidative metabolism has rarely been studied. In this study, we have investigated the significance of bicarbonate transport in osteoclasts. We found that osteoclasts possess several potential proteins for bicarbonate transport, including carbonic anhydrase IV and XIV, and an electroneutral bicarbonate co-transporter NBCn1. We have also shown that inhibiting the function of these proteins has a significant impact on bone resorption and osteoclast morphology. Furthermore, we have explored oxidative metabolism in chondrocytes and found that carbonic anhydrase III (CA III), a protein linked to the prevention of protein oxidation in muscle cells, is also present in mouse chondrocytes, where its expression correlates with the presence of reactive oxygen species. Thus, our study provides novel information on the regulation of cellular metabolism in calcified tissues.

Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

Potential of the Osteoclast’s Proton Pump as a Drug Target in Osteoporosis

Decreasing bone mass during aging predisposes to fractures and it is estimated that every second woman and one in five men will suffer osteoporotic fractures during their lifetime. Bone is an adaptive tissue undergoing continuous remodeling in response to physical and metabolic stimuli. Bone mass decreases through a net negative balance in the bone remodeling process of bone, in which the new bone incompletely replaces the resorbed bone mass. Bone resorption is carried out by the osteoclasts; the bone mineral is solubilized by acidification and the organic matrix is subsequently degraded by proteases. Several classes of drugs are available for prevention of osteoporotic fractures. They act by different mechanisms to increase bone mass, and some of them act mainly as antiresorptives by inhibition of osteoclast formation or their function. Optimally, a drug should act selectively on a specific process, since other processes affected usually result in adverse effects. The purpose of this study was to evaluate whether the osteoclastic vacuolar adenosine trisphosphatases (V-ATPase), which drives the solubilization of bone mineral, can be selectively inhibited despite its ubiquitous cellular functions. The V-ATPase is a multimeric protein composed of 13 subunits of which six possesses two or more isoforms. Selectivity for the osteoclastic V-ATPase could be provided if it has some structural uniqueness, such as a unique isoform combination. The a3 isoform of the 116kDa subunit is inevitable for bone resorption; however, it is also present in, and mainly limited to, the lysosomes of other cells. No evidence of a structural uniqueness of the osteoclastic V-ATPase compared to the lysosomal V-ATPase was found, although this can not yet be excluded. Thus, an inhibitor selective for the a3 isoform would target the lysosomal V-ATPase as well. However, the results suggest that selectivity for bone resorption over lysosomal function can be obtained by two other mechanisms, suggesting that isoform a3 is a valid target. The first is differential compensation; bone resorption depends on the high level of a3 expression, and is not compensated for by other isoforms, while the lower level of a3 in lysosomes of other cells may be partly compensated for. The second mechanism is because the bone resorption process itself is fundamentally different from lysosomal acidification because of the chemistry of bone dissolution and the anatomy of the resorbing osteoclast. By this mechanism, full inhibition of bone resorption is obtained with more than tenfold lower inhibitor concentration than those needed to fully inhibit lysosomal acidification. The two mechanisms are additive. Based on the results, we suggest that bone resorption can be selectively inhibited if VATPase inhibitors that are sufficiently selective for the a3 isoform over the other isoforms are developed.

Bone Health, Osteoporosis and Fracture Risk in Neurofibromatosis 1 - An Emphasis on Osteoclasts

Neurofibromatosis 1 (NF1) is an autosomal dominant hereditary syndrome, affecting skin, neural tissues and skeleton. Hallmarks of NF1 include benign cutaneous neurofibroma tumors, pigmentation lesions on the skin and in the iris, learning disabilities and predisposition to selected malignancies. Low bone mineral density (BMD) and osteopenia/osteoporosis are common in NF1. Osteoporosis is a systemic disorder characterized by low bone mineral density and increased fracture risk. Treatment of osteoporosis aims to prevent falls and decrease fracture risk. Osteoporosis is diagnosed in adults by measuring BMD and evaluating clinical risk factors of the patient. Bone turnover is a process of old bone resorbed by osteoclasts and new bone formed by osteoblasts. Multinuclear osteoclasts are derived from osteoclast progenitors, which can be isolated from peripheral blood. Osteoclast progenitors were isolated from 17 NF1 patients and healthy controls, and cultured in vitro to osteoclasts. NF1 osteoclasts are hyperactive, displaying increased differentiation and resorption capacity, abnormal morphology and tolerance to serum deprivation compared to control osteoclasts. These findings expanded the study to evaluate the effects of bisphosphonates, drugs designed to treat osteoporosis, in osteoclasts derived from blood samples of 20 NF1 and control persons. The number of control osteoclasts was expectedly reduced after bisphosphonate treatment. However, NF1 osteoclasts tolerated the apoptotic effect of alendronate, zoledronic acid and clodronate in vitro compared to controls. NF1-related osteoporosis was found in ~20 % of the patients, and selected laboratory parameters were measured. Patients with NF1 have increased levels of serum CTX and PINP, reflecting increased bone turnover in vivo. BMD decreases progressively in NF1 as evaluated in 19 NF1 patients 12 years after their initial BMD measurement. Patients with NF1-related osteopenia often progress to osteoporosis. This was found in patients aged 37-76.

Bioactive Glass in Lumbar Spondylodesis. A Pre-Clinical and Clinical Study

Bioactive glasses (BGs) form a group of synthetic, surface-active, composition-dependent, silica-based biomaterials with osteoconductive, osteopromotive, and even angiogenic, as well as antibacterial, properties. A national interdisciplinary research group, within the Combio Technology Program (2003–2007), developed a porous load-bearing composite for surgical applications made of BG 1–98 and polymer fibers. The pre-clinical part of this thesis focused on the in vitro and in vivo testing of the composite materials in a rabbit femur and spinal posterolateral fusion model. The femur model failed to demonstrate the previously seen positive effect of BG 1–98 on osteogenesis, probably due to the changed resorption properties of BG in the form of fibers. The spine study was terminated early due to adverse events. In vitro cultures showed the growth inhibition of human mesenchymal stems next to BG 1–98 fibers and radical pH changes. A prospective, long-term, follow-up study was conducted on BG–S53P4 and autogenous bone used as bone graft substitutes for instrumented posterolateral spondylodesis in the treatment of degenerative spondylolisthesis (n=17) and unstable burst fractures (n=10) during 1996–1998. The operative outcome was evaluated from X-rays and CT scans, and a clinical examination was also performed. On the BG side, a solid fusion was observed in the CT scans of 12 patients, and a partial fusion was found in 5 patients, the result being a total fusion rate in all fusion sites (n=41) 88% for levels L4/5 and L5/S1 in the spondylolisthesis group. In the spine fracture group, solid fusion was observed in five patients, and partial fusion was found in five resulting in a total fusion rate of 71% of all fusion sites (n=21). The pre-clinical results suggest that under certain conditions the physical form of BG can be more critical than its chemical composition when a clinical application is designed. The first long-term clinical results concerning the use of BG S53P4 as bone graft material in instrumented posterolateral spondylodesis seems to be a safe procedure, associated with a very low complication rate. BG S53P4 used as a stand-alone bone substitute cannot be regarded as being as efficient as AB in promoting solid fusion.

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

Vesicular Trafficking in Osteoclasts

Osteoclasts are multinucleated bone-degrading cells that undergo large changes in their polarisation and vesicular trafficking during the bone resorption cycle. Rab proteins are small GTPases that offer both temporal and spatial regulation to the transport between membranous organelles. Previously the presence and function of only few of the currently known 60 Rab proteins in osteoclasts have been reported. In this study, the expression of 26 Rab genes in bone-resorbing osteoclasts was demonstrated with gene-specific primer pairs. The further analysis of three Rab genes during human osteoclast differentiation revealed that Rab13 gene is highly induced during osteoclastogenesis. The presence of Rab13 protein in the secretory vesicles directed towards the ruffled border and in the endocytotic or transcytotic pathways in resorbing osteoclasts was excluded. The localisation of Rab13 suggests that that it is associated with a previously unknown vesicle population travelling between the trans-Golgi network and the basolateral membrane in bone resorbing osteoclasts. Rab proteins convey their functions by binding to specific effector proteins. We found a novel Rab13 interaction with endospanins-1 and -2 that are yet poorly characterised small transmembrane proteins. The Rab13 subfamily member Rab8 also bound to endospanins, while Rab10 and unrelated Rabs did not. Rab13 and endospanin-2 co-localised in perinuclear vesicles in transfected cells, demonstrating the interaction also in vivo. The inhibition of Rab13 did not interfere with the localisation of endospanin-2 nor did it affect the cell surface expression of growth hormone receptor, as has been previously described for endospanins. The physiological role of this novel protein-protein interaction thus remains to be clarified. The analysis of the transcytotic route in bone resorbing osteoclasts revealed that multiple vesicle populations arise from the ruffled border and transport the bone degradation products for exocytosis. These vesicles are directed to the functional secretory domain that is encircled by an actin-based molecular barrier. Furthermore, the transcytotic vesicles contain abundant Helix pomatia lectin binding sites and represent lipid raft concentrates. Finally, autophagosomal compartments may also be involved in the transcytosis.

Analysis of Alpha CA Genes in Early Vertebrates and ; High-Invertebrates

Carbonic anhydrases are enzymes that are ubiquitously found in all organisms that are engaged in catalyzing the hydration of carbon dioxide to form bicarbonate and proton and vice versa. They are crucial in the process of respiration, bone resorption, pH regulation, ion transport, and photosynthesis in plants. Out of the five classes of carbonic anhydrase α, β, γ, δ, ζ this study focused in the α carbonic anhydrases. This class of CAs constitute of 16 subfamilies in mammals that include 3 non-active enzymes known as Carbonic Anhydrase Related Proteins. The inactiveness of these enzymes is due to the loss of one or more Histidine residues in the active site. This thesis was conducted based on the aim of studying evolutionary analysis of carbonic anhydrase sequences from organisms spanning from the Cambrian age. It was carried out in two phases. The first phase was the sequence collection, which involved many biological sequence databases as a source. The scope of this segment included sequence alignments and analysis of the sequence manually and in an automated form incorporating few analysis tools. The second Phase was phylogenetic analysis and exploring the subcellular location of the proteins, which was key for the evolutionary analysis. Through the medium of the methods conducted with respect to the phases mentioned above, it was possible to accomplish the desired result. Certain thought-provoking sequences were come across and analyzed thoroughly. Whereas, Phylogenetics showed interesting results to bolster previous findings and new findings as well which lay bedrock for future intensified studies.