Difference Between Collagen and Elastin | Collagen vs Elastin Home / Science & Nature / Science / Biology / Difference Between Collagen and Elastin Difference Between Collagen and Elastin Posted on Collagen vs Elastin Connective tissues are important for binding and connecting other tissues within the body. They also provide strength, support, and shape to the tissues. The connective tissue is a system in which cells are scattered throughout an extracellular matrix. In addition to cells, the insoluble protein fibers are also embedded in the matrix. The matrix is called the ground substance. These tissues are widely distributed in the body, matrix bone, tendons and ligaments , and cartilage . The connective tissues consist of four basic tissues, collagen, elastin, proteoglycans, and glycoproteins. Source: Ruth Lawson, Otago Polytechnic, en.wikibooks Collagen Collagen is the most abundant protein found in connective tissues. It provides great tensile strength and holds cells together. In addition, it helps to align the cells, thus allows in proliferation and differentiation of cells. Tropocollagen is the basic structural unit of collagen that consists of α- chains.

Each α-chain is made up of three polypeptide chains twisting around each other in a triple helix to form a rope like structure. There are hydrogen bonds between the polypeptide chains to hold them tightly. Each of this polypeptide chain has equal length and contains about 1000 amino acids residues. Various triple helical combinations of polypeptides in α-chains result multiple types of collagen in humans connecting tissues (19 types of collagen have been identified so far). Most abundant types of collagen are distributed in skin, tendon, bone, comea, articular cartilage, intervertebral disk, fetal skin, cardiovascular system, placenta etc. The cross-links are important to provide a high tensile strength in collagen. There are three types of inter or intramolecular cross-links involved to stabilize the collagen fibers; they are aldol condensation, Schiff base, and lysinonorleucine. Source: wikicommons Elastin Elastin is made up of basic subunit called tropoelastin, which contains about 800 amino acid residues. The cross-links of elastin are more complex than that of collagens. Desmosine is the major type of cross-links found in elastin.

They are formed from the condensation of the allysine residues with lysine. Elastin often occurs with collagen in connective tissues. It is a rubber-like protein so that it can stretch up to several times their length and return back to their original shape and length when the tension is released. Due to this property, it is largely found in the tissues associated with lungs , blood vessels and ligaments, which undergo large expansions. In addition, they are also found in places like skin, ear cartilage and several other tissues in small quantities. What is the difference between Collagen and Elastin? • There is only one genetic type of elastin, whereas there are many different genetic types of collagen. • Elastin has enough capacity to stretch and subsequently to recoil while collagen has no such capacity to stretch. • Collagen’s primary structure has repeating (Gly-X-Y) sequences whereas, in elastin, there are no such repeating (Gly-X-Y) sequences. • In contrast to collagen, there is no formation of triple helix in elastin. • Hydroxylysine is present in collagen, whereas it is absent in elastin.

• Glycosylated hydroxylysine is present in collagen, whereas it is absent in elastin. • The major cross-links formed in collagen are intramolecular aldol cross-links, whereas those in elastin are intramolecular desmosine cross-links. Related posts: Definition of Elastin Definition of Elastin Causes of a Heart Attack Slideshow Elastin: A protein that coil and recoils like a spring within the elastic fibers of connective tissue and accounts for the elasticity of structures such the skin, blood vessels, heart, lungs, intestines, tendons, and ligaments. Elastin functions in connective tissue together with collagen. Whereas elastin provides elasticity, collagen provides rigidity to connective tissue. Elastin is normally no longer made after puberty and aging begins. Also called elasticin. Last Editorial Review: 5/13/2016 Proteins Examples Proteins Examples Proteins A protein is a polymer macromolecule, meaning it's built from long chains of amino acids.

These chains, which also include hydrogen, oxygen, carbon, and nitrogen, usually contains between 200 and 300 amino acids, although the peptides are much smaller and the titins are much larger. Titins are so large, in fact, that they can contain over 33,000 amino acids. It's important to not confuse the protein that we eat with the proteins that support cellular function. A functions of every living cell rely on protein to make it happen, and even though eating a juicy steak is tasty, it's not the same thing as supporting cellular life. There are three types of proteins: fibrous, globular, and membrane. Each type serves different functions and has a different makeup, but they are all built on the same components that form all proteins. Examples of Proteins: 1. Fibrous Proteins Fibrous proteins are just what they sound like. They are composed of long strings of individual fibers the bundle together to form a more solid "wall" of protein. These bundles form the muscles, tendons, bones, and other connective tissues in animals. These specific proteins include actin, collagen, elastin, keratin, myosin, tubulin, and many more. 2.

Globular Proteins Globular proteins are easy to remember because the word sounds like, "glob," and a glob of water is all it takes to break down these proteins. They are far more readily water soluble than the other types of proteins, which is helpful because their job is to transport and regulate substances. Some of the globular proteins are albumins, alpha globulin, carboxypeptidase, ependymin, fibrin, hemoglobin, integrin, myoglobin, selectin, thrombin, and von willebrand factor. 3. Membrane Proteins These proteins get their name from their specific jobs in which they interact with the cell membranes within the organism when they serve as transporters for molecules, a bridge that allows interactions between two cells, or when they relay signals within the cell. Membrane proteins are interesting because more than half of the pharmaceuticals on the market today work by affecting the membrane proteins in some way, and because scientists believe that between 20% and 30% of all the genes in the genome work to encode the membrane proteins. That makes them pretty important to cellular function, and therefore, life itself.

Some of these membrane proteins include hormone receptors, hydrolases, transferases, and many more. Related Links: Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis. - PubMed - NCBI J Biomed Mater Res A. 2015 Mar;103(3):1218-30. doi: 10.1002/jbm.a.35257. Epub 2014 Aug 28. Human elastin polypeptides improve the biomechanical properties of three-dimensional matrices through the regulation of elastogenesis. 1Department of Health Sciences, University of Piemonte Orientale "A. Avogadro", 28100, Novara, Italy. Abstract The replacement of diseased tissues with biological substitutes with suitable biomechanical properties is one of the most important goal in tissue engineering. Collagen represents a satisfactory choice for scaffolds. Unfortunately, the lack of elasticity represents a restriction to a wide use of collagen for several applications. In this work, we studied the effect of human elastin-like polypeptide (HELP) as hybrid collagen-elastin matrices.

In particular, we studied the biomechanical properties of collagen/HELP scaffolds considering several components involved in ECM remodeling (elastin, collagen, fibrillin, lectin-like receptor, metalloproteinases) and cell phenotype (myogenin, myosin heavy chain) with particular awareness for vascular tissue engineering applications. Elastin and collagen content resulted upregulated in collagen-HELP matrices, even showing an improved structural remodeling through the involvement of proteins to a ECM remodeling activity. Moreover, the hybrid matrices enhanced the contractile activity of C2C12 cells concurring to improve the mechanical properties of the scaffold. Finally, small-angle X-ray scattering analyses were performed to enable a very detailed analysis of the matrices at the nanoscale, comparing the scaffolds with native blood vessels. In conclusion, our work shows the use of recombinant HELP, as a very promising complement able to significantly improve the biomechanical properties of three-dimensional collagen matrices in terms of tensile stress and elastic modulus. © 2014 Wiley Periodicals, Inc.

C2C12; collagen scaffold; extracellular matrix remodeling; human elastin-like polypeptide; tissue engineering PMID: Keratin - Biology-Online Dictionary Definition noun, plural: keratins One of the fibrous structural proteins, and is a constituent of hair , nails , skin , feathers , hooves, horns , etc. Supplement Scleroproteins or the fibrous proteins are one of the three major types of proteins; the other two are spheroproteins and membrane proteins . Scleroproteins are characterized by their long protein filaments. They act as structural proteins. They are usually water-insoluble. Some of the scleroproteins are keratin, collagen , elastin , and fibroin. Keratins are fibrous structural proteins that constitute various biological structures such as hair, nails, skin, feathers, hooves, horns, etc. They are made up of coiled polypeptide chains and when they combine they form supercoils. Keratins protect epithelial cells from damage. Skin cells that are constantly exposed to pressure and rubbing leads to the formation of calluses. The skin thickens and the epidermal cells undergo cornification.

Cornification is a process in which a keratinized layer of epidermis forms and serves as an epidermal barrier. During cornification, keratin fills up the cell resulting in the loss of cytoplasmic organelles and the cessation of metabolism. Ultimately, the fully keratinized cells undergo a programmed cell death. Word origin: Greek keras (horn) + -in See also: collagen; myosin; ( )keratin; fibroin /elastin / silk - BIOLOGY - Bio 100 Collagen; myosin; ( )keratin; fibroin /elastin / silk SCHOOL View Full Document This is the end of the preview. Sign up to access the rest of the document. Unformatted text preview: collagen; myosin; ( )keratin; fibroin /elastin / silk protein in insects and spiders; Reject fibrinogen. Award [1 max] for example of globular proteins.

8 (b) polar amino acids are hydrophilic and non-polar amino acids are hydrophobic; position of polar and non-polar amino acids determine protein shape / function / location; (in channel proteins) hydrophilic amino acids line the channels and allow transport of ions/polar substances; non-polar amino acids are in contact/embedded within the lipid membrane; polar amino acids on the surface proteins make them water soluble; non-polar in the centre of water-soluble proteins stabilize the structure; 3 max Accept any of the above points if clearly explained using a suitably labelled diagram. 9. two polymers shown; arranged in a double helix; sugar shown connected to base; sugar-phosphate backbone shown; If only one nucleotide is drawn, award [2 max] sugar identified as deoxyribose; hydrogen bonding between bases shown; diagram shows complementary base pairing / A bonded to T, C with G; Award previous mark if bases (unlabelled) are shown in the diagram but the complementary base pairing is explained in the annotation. covalent bonding between phosphate and sugar; 5 max (Remember, up to TWO 'quality of construction' marks per essay) . 10.

RNA DNA ribose deoxyribose; (normally) single stranded double stranded; uracil thymine; no double helix helix; N.B . Histone proteins are only in eukaryotic DNA not prokayotic.... Highland Park High, Highland Park BIOLOGY Bio 100 - Spring 2011 CellularRespirationTest 1. KEY 24December2008 HowmanyATPmolecules(netyield)areproduce Cellular Respiration Test Key What Are Collagen and Elastin? | eHow What Are Collagen and Elastin?  Email Save Collagen and elastin are structural proteins made and used in the human body. Collagen is found primarily in tendons, ligaments, and the connective tissue of skin, blood vessels, and lungs. Elastin is found primarily in the artery walls, lungs, intestines, and skin. These proteins work in partnership in connective tissues. Function Collagen gives connective tissue and organs rigidity so that they can function, and elastin lets them stretch and return to their original state. Collagen does not allow the elastin to stretch to the point of breaking.

In the skin, collagen and elastin are the primary components of the dermis--the layer right beneath the epidermis. They provide the support structure of the skin. Significance The human body stops producing elastin when you hit puberty. This is when the aging process begins. Without elastin replenishment, collagen begins to lose its elasticity and begins to weaken. The loss of the skin's tone and resiliency are the most common visual markers of this process. Facial expressions begin to put stress on collagen and after a period of time, wrinkles begin to appear. Misconceptions Some companies advertise the use of collagen and elastin in their anti-aging creams and moisturizers; however, these products work only on the skin surface. According to the contributing dermatologists at MedicineNet, creams and moisturizers cannot penetrate the skin and are not designed to be absorbed; therefore they will not undo the cumulative effect of collagen loss. The creams slow the rate of water loss and keep the skin supple. Injectable Fillers Collagen can be used as an anti-aging agent in the form of an injectable filler. The filler is placed right beneath the surface of the skin.

This collagen replenishes the support structure of the skin by supplementing existing collagen and restoring the contour of the skin. Injectable fillers will smooth facial lines and most scars. Warning The fillers may cause allergic reactions and a skin test prior to the first treatment is recommended. Additionally, slight bruising, puffiness, redness and tenderness may occur around the injection site. Certain medical conditions may rule out the use of fillers. Proteins |What are Proteins | Characteristics of Proteins | Biology@TutorVista.com Top Proteins Proteins are important molecules in cells. Proteins are the major component of the dry weight of cells. The name protein is derived from a Greek word Proteios which means pre-eminent or first. This name was fist suggested in 1838 by a Swedish chemist Berzelius. He suggested it to a Dutch chemist Mulder and he referred it to the complex organic substances found in the cells of living beings. Proteins are the most abundant intracellular macro-molecules. Proteins are connected intimately with all chemical and physical activity, which constitutes the life of the cell. Proteins are present in and vital to all living cells.

They provide structure, protection to the body of multicellular organism in the form of skin, hair, callus, cartilage, ligaments, muscles, tendons. Proteins regulate and catalyze the body chemistry in the form of hormones, enzymes, immunoglobulins etc. Back to Top Proteins are known as building blocks of life. Proteins are biomolecules, usually large in size, that consists of one or more chains of amino acids. Proteins perform variety of functions like catalyzing metabolic reactions, replication of DNA, response to stimuli, and transporting molecules. Proteins differ from each other mainly in the sequences of amino acids. Characteristics of Proteins General Characteristics of Proteins are as follows: Proteins are organic substances, they are made up of nitrogen and also, oxygen, carbon an d hydrogen. Proteins are the most important biomolecules, they are the fundamental constituent of the cytoplasm of the cell. Proteins are the structural elements of body tissues. Proteins are made up of amino acids. Proteins gives heat and energy to the body and also aid in building and repair. Only small amounts of proteins are stored in the body as they can be used up quickly on demand.

Proteins are considered as the bricks, they make up bones, muscles, hair and other parts of the body. Proteins like enzymes are functional elements that take part in metabolic reactions. Antibodies, blood haemoglobin are also made of proteins. Proteins have a molecular weight of 5 to 300 kilo-daltons. Properties of Proteins The general properties of proteins are similar to those of the amino acids: Physical Properties of Proteins Classification of Proteins Based on Shape They are grouped under two categories globular and fibrous. Globular or Corpuscular Proteins Globular proteins have axial ratio less than 10. They are compactly folded and coiled. and possess a relatively spherical or ovoid shape. They are usually soluble in water and in aqueous media. Example Insulin, plasma albumin, globulin enzymes. Fibrous or Fibrillar Proteins These proteins have axial ratio more than 10, hence, they resemble long ribbons or fibres in shape. They are mostly found in animals, and are not soluble in water or in solution of dilute acids. Fibrous proteins aid in protection and structural support.

Example: Collagen, Keratin, Elastins, Fibroin. Classification of Proteins Based on Composition and Solubility Simple Proteins or Holoproteins These proteins are made of only one type of amino acid, as structural component, on decomposition with acids, they liberate constituent amino acids. They are mostly globular type of proteins except for scleroproteins, which are fibrous in nature. Simple proteins are further classified based on their solubility. Protamines and histones - These proteins occur only in animals and are basic proteins. The possess simple structure and low molecular, are water soluble and are not coagulated by heat. The are strongly basic in character due to the high content of lysine, arginine. Example: Protamines - salmine, clupine, cyprinine; Histones - nucleoshistones, globin. Albumins - They are widely distributed in nature, mostly seen in seeds. They are soluble in water and dilute solutions of acids, bases and salts. Example: Leucosine, legumeline, serum albumin.

Globulins - They are of two types, pseudoglobulins which are soluble in water, other is euglobulins which are insoluble in water. They are coagulated by heat. Example: Pseudoglobulin, serum globulin, glycinine. etc. Scleroproteins or Albuminoids - These occur mostly in animals and are commonly known as animal skeleton proteins, they are insoluble in water, and in dilute solution of acids, based and salts. Conjugated or Complex Proteins or Heteroproteins These are proteins that are made of amino acids and other organic compounds. The non-amino acid group is termed as prosthetic group. Complex proteins are further classified based on the type of prosthetic group present. Metalloproteins - These are proteins linked with various metals. Example: casein, collagen, ceruloplasmin, etc. Chromoproteins - These are proteins that are coupled with a colored pigment. Example: Myoglubin, hemocyanin, cytochromes, flavoproteins, etc. Glycoproteins and Mucoproteins - These proteins contain carbohydrates as the prosthetic group.

Example: Glycoproteins - egg albumin, serum globulins, serum albumins; Mucoproteins - Ovomucoid, mucin etc. Phosphoproteins - These proteins are linked with phosphoric acid. Example: casein. Lipoproteins - Proteins forming complexes with lipids are lipoproteins. Example: lipovitellin, lipoproteins of blood. Nucleoproteins - These are compounds containing nucleic acids and proteins. Example: Nucleoproteins, nucleohistones, nuclein. Derived Proteins Back to Top Below is the list of proteins functions. Proteins are seen in muscles, hair, skin and other tissues, they constitute the bulk of body's non-skeletal structure. Example: The protein keratin is present in nails and hair. Some proteins are hormones and regulate many body functions. Example: Insulin hormone is a protein and it regulated the blood sugar level. Some proteins act enzymes, they catalyze or help in biochemical reactions. Example: Pepsin and Tripsin. Some proteins act as antibodies, they protect the body from the effect of invading species or substances.

Proteins transport different substances in blood of different tissues. Example: Haemoglobin is a oxygen transport protein. Contractile proteins help in contraction of muscle and cells of our body. Example: Myosin is contractile protein. Fibrinogen a glycoprotein helps in healing of wounds. It prevents blood loss and inhibits passage of germs. Types of Proteins A Keratin Peptide Inhibits Mannose-Binding Lectin | The Journal of Immunology Follow The Journal of Immunology on RSS A Keratin Peptide Inhibits Mannose-Binding Lectin Michael C. Montalto, Charles D. Collard, Jon A. Buras, Wende R. Reenstra, Rebecca McClaine, David R. Gies, Russell P. Rother and Gregory L. Stahl J Immunol March 15, 2001, 166 (6) 4148-4153; DOI: Michael C.

Montalto Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; Search for this author on this site Charles D. Collard Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; Search for this author on this site Rebecca McClaine Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; Search for this author on this site Gregory L. Stahl Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; PDF Abstract Complement plays a significant role in mediating endothelial injury following oxidative stress.

We have previously demonstrated that the lectin complement pathway (LCP), which is initiated by deposition of the mannose-binding lectin (MBL), is largely responsible for activating complement on endothelial cells following periods of oxidative stress. Identifying functional inhibitors that block MBL binding will be useful in characterizing the role of the LCP in disease models. The human cytokeratin peptide SFGSGFGGGY has been identified as a molecular mimic of N-acetyl-d-glucosamine (GlcNAc), a known ligand of MBL. Thus, we hypothesized that this peptide would specifically bind to MBL and functionally inhibit the LCP on endothelial cells following oxidative stress. Using a BIAcore 3000 optical biosensor, competition experiments were performed to demonstrate that the peptide SFGSGFGGGY inhibits binding of purified recombinant human MBL to GlcNAc in a concentration-dependent manner. Solution affinity data generated by BIAcore indicate this peptide binds to MBL with an affinity (KD) of 5 × 10−5 mol/L.

Pretreatment of human serum (30%) with the GlcNAc-mimicking peptide (10–50 μg/ml) significantly attenuated MBL and C3 deposition on human endothelial cells subjected to oxidative stress in a dose-dependent manner, as demonstrated by cell surface ELISA and confocal microscopy. Additionally, this decapeptide sequence attenuated complement-dependent VCAM-1 expression following oxidative stress. These data indicate that a short peptide sequence that mimics GlcNAc can specifically bind to MBL and functionally inhibit the proinflammatory action of the LCP on oxidatively stressed endothelial cells. The lectin complement pathway (LCP) 4 is an important mediator of the innate immune response, especially during early childhood ( 1 , 2 ). The LCP is typically initiated by the binding of mannose-binding lectin (MBL) to carbohydrate moieties present on the surface of various bacteria, yeast, and other pathogens ( 2 ).

Bound MBL associates with the C1r2C1s2-like MBL-associated serine proteases (MASP-1 and MASP-2) which then cleave C2 and C4 to form the classical C3 convertase ( 3 , 4 ). The LCP converges with the Ab-dependent classical pathway and Ab-independent alternative pathway at the level of C3 cleavage ( 5 ). We have previously shown that the LCP is activated on human endothelial cells that have been subjected to oxidative stress ( 6 ). Furthermore, we observed MBL deposition on rat myocardium following ischemia-reperfusion, suggesting that MBL may be proinflammatory during periods of oxidative stress in vivo ( 6 ). Additionally, MBL has been implicated in the development of other proinflammatory conditions such as IgA nephropathy and Henoch-Schönlein purpura nephritis ( 7 , 8 , 9 ). Therefore, the development and characterization of potential inhibitors of MBL may be beneficial to understanding the role of MBL as an inflammatory mediator.

MBL is a C-type lectin (collectin) that has a binding affinity for N-acetyl-d-glucosamine (GlcNAc) and mannan ( 10 , 11 , 12 , 13 ). In humans, MBL forms an oligomeric structure composed of subunit monomers (32 kDa) arranged in a hexamer of trimers ( 13 , 14 ). Association of the N-terminal collagen region of multiple monomers creates a clustering of the C-terminal carbohydrate recognition domains (CRD) ( 12 , 14 ). This bouquet-like conformation allows for the binding of multiple sugar carbohydrate residues and likely contributes to the high binding affinity of MBL for oligosaccharides and bacteria cell walls ( 5 , 12 ). Although the LCP can be inhibited by natural ligands such as GlcNAc or mannose, carbohydrates serve as poor potential therapeutics due to their limited bioavailability ( 15 ). For this reason, our laboratory has focused on identifying alternative inhibitors of MBL and the LCP. Shikham et al.

( 16 ) have demonstrated that mouse and human Abs specific for GlcNAc cross-react with the cytokeratin 14 decapeptide SFGSGFGGGY. Additionally, it was shown that several GlcNAc-specific lectins, including Datura stramonium lectin, Lycopericon esculentum lectin, Solanum tuberosum lectin, wheat germ agglutinin, and Ulex europaeus lectin II, were capable of binding this peptide sequence ( 16 ). More recently, another group demonstrated that the enamel matrix protein amelogenin, which is capable of binding GlcNAc, could also bind the peptide SFGSGFGGGY, confirming that this sequence is a molecular mimic of GlcNAc ( 17 ). Since MBL has a natural binding affinity for GlcNAc, we hypothesized that the GlcNAc-mimicking peptide SFGSGFGGGY would specifically bind MBL and inhibit the LCP on oxidatively stressed endothelial cells. Thus, this is the first report of a noncarbohydrate ligand interacting with the CRD of MBL.

Materials and Methods HUVECs HUVECs were isolated and cultured as previously described ( 18 ). Briefly, HUVECs were harvested with 0.1% collagenase (Worthington Biochemical, Lakewood, NJ) and suspended in medium 199 containing 20% heat-inactivated bovine calf serum (Life Technologies, Rockville, MD). The cells were grown in a 95% air/5% CO2 incubator at 37°C. Endothelial cell purity was assessed as previously described ( 18 ). HUVECs were used at passages 1–3. Production of mAbs Production and characterization of the anti-human MBL mAb 3F8 has been previously described ( 6 ). An anti-porcine C5a mAb was used as an isotype control ( 19 ). Cloning and expression of recombinant human MBL Human recombinant MBL (rMBL) was cloned by PCR amplification of human liver cDNA. PCR primers were synthesized by Life Technologies.

The 5′ primer (GCGCGGATCCACCATGGCCCTGTTTCCATCACTCCC) was generated from the 5 ′ end of the human MBL (hMBL) coding sequence and contains an upstream BamHI site followed by a Kozak consensus sequence ( 20 ). Base 4 of the coding region was changed from a thymine to a guanine, resulting in a serine to alanine change to optimize ribosome binding. The 3′ primer (CCTACCTGCAGGTCAGATAGGGAACTCACAGACG) was generated from the 3 ′ end of the hMBL coding sequence and contains an Sse8337 cloning site. PCR was performed using the Advantage PCR kit with human liver QUICK-Clone cDNA as the template using the manufacturer’s protocol (Clontech, Palo Alto, CA). The resulting 771-bp band was cloned into the TA cloning vector pCR2.1-TOPO (Invitrogen, Carlsbad, CA) and the sequence was verified.

The fragment was directionally subcloned into the mammalian expression vector APEX-3P that was digested with BamHI/Sse8337, resulting in the plasmid pAPEX-3P hMBL ( 21 ). pAPEX-3P hMBL was transfected into 293 EBNA human embryonic kidney cells (Invitrogen) using the transfectam lipid transfection reagent (Promega, Madison, WI) as previously described ( 21 ). Cells were selected in growth medium (DMEM with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mmol/L glutamine, and 250 U/ml G418) containing puromycin at 1 μg/ml for 7–10 days. A pooled cell population was tested by ELISA for the presence of recombinant hMBL and expanded for production.

Confluent T-175 flasks of selected cells were washed with PBS before the addition of 30 ml of HB Pro serum-free medium (Irvine Scientific, Santa Ana, CA) containing 250 U/ml G418 and 1 μg/ml puromycin. Supernatants were harvested on two consecutive 4-day intervals, at which time cell debris was removed by centrifugation. Cleared supernatants were stored at 4°C before purification. hMBL was purified from cell supernatants as previously described with some modifications ( 22 ). Briefly, hMBL-containing supernatants (1 liter) were adjusted to 10 mmol/L CaCl2 and rocked overnight at 4°C with 30 ml of mannan-agarose (Sigma, St. Louis, MO) previously washed with TBS (150 mmol/L NaCl, 20 mmol/L Tris, pH 7.4). Following batch binding, the resin was allowed to settle and supernatant was removed.

The mannan-agarose resin was transferred to a 100-ml chromatography column (Bio-Rad, Hercules, CA) and washed with 5 volumes of TBS supplemented with 10 mmol/L CaCl2. The hMBL was then eluted with TBS containing 10 mmol/L EDTA. One-milliliter fractions were collected and samples with significant absorbances at A280 were pooled and dialyzed against PBS/0.5 mol/L NaCl (pH 7.4). Protein was concentrated in a Centriprep 30 concentrator (Amicon, Beverly, MA) and sterile filtered using a 0.22-μm Millipore syringe filter (Millipore, Bedford, MA). PAGE analysis of hMBL was performed under reducing and nonreducing conditions. Five micrograms of purified hMBL was separated on a 4–20% Novex gradient gel (Invitrogen) and proteins were stained with Coomassie blue. High order oligomers of MBL were observed under nonreducing conditions and a single monomer (∼30 kDa) was observed under reducing conditions (data not shown).

For control experiments, native hMBL was purified from human serum as previously described ( 6 ). Conjugation of GlcNAc to BSA GlcNAc-phenylisothiocyanate (5 mg; Sigma) was dissolved in 5 ml of DMSO and mixed with 3 ml of BSA (5 mg/ml in 0.1 mol/L Na2CO3 buffer, pH 9.0) and rotated overnight at 4°C. The resulting BSA-GlcNAc conjugate was dialyzed against 100 volumes of 0.1 mol/L Tris-HCl (pH 7.0) for 4 h and further dialyzed against PBS for 48 h. A mock control reaction was performed in parallel using DMSO containing no GlcNAc-phenylisothiocyanate. BSA-GlcNAc conjugation was confirmed by ELISA using an anti-O-linked GlcNAc mAb (Affinity Bioreagents, Golden, CO) (data not shown).

Surface plasmon resonance and solution affinity kinetics The peptide SFGSGFGGGY (referred to here as Glupep4 for N-acetyl-d-glucosamine peptide) was synthesized by the Dana-Farber Molecular Biology core facility (Boston, MA). Binding analysis was performed using a BIAcore 3000 and BIAevaluation 3.0.2 software (BIAcore, Uppsala, Sweden). A biosensor C1 chip (containing no dextran) was washed with 0.1 mol/L glycine-NaOH/0.3% Triton X-100 and activated with N-hydroxysuccinimide and N-ethyl-N′-(dimethyl-aminopropyl)-carbodiimide according to the manufacturer’s instructions. GlcNAc-BSA was diluted to 100 μg/ml in acetate buffer (pH 4.0) and injected manually over a single flow cell until a resonance unit value of ∼1000 was obtained. BSA from the mock conjugation reaction was immobilized on a control flow cell and used to subtract nonspecific binding and bulk changes in the refractive index.

To generate a calibration curve for solution affinity analysis, rMBL was diluted in running buffer (143 mmol/L NaCl, 10 mmol/L HEPES, 40 mmol/L CaCl2, 40 mmol/L MgCl2, pH 7.4) and injected at 20 μl/min for 3 min. Each concentration was separated with 2.5 min of disassociation time and a 30-s regeneration with 100 mmol/L glycine (pH 3.0). Each sensogram represents the relative response after subtraction of the reference (control) flow cell. The initial slope of the association phase is directly proportional to concentration under mass transport limited conditions only ( 23 ). Therefore, we confirmed rMBL binding to be mass transport limited by varying the flow rate on separate injections (data not shown). A calibration curve was generated by plotting the concentration of rMBL against the initial slope of each response at 20 s after injection.

For inhibition analysis and KD calculations, varying concentrations of Glupep (100–0 μg/ml; 1 μg/ml of Glupep = 1.069 μmol/L), an inhibitory mAb specific for human MBL (3F8, 5 or 50 μg/ml) or an isotype control Ab (50 μg/ml) were incubated with a constant concentration of rMBL (6.9 nmol/L in running buffer) for 15–30 min at 23°C. Flow rate, contact time, and regeneration conditions for Glupep inhibition experiments were identical to conditions used for standard curve generation. For control experiments, a 90-s contact time was used. BIAevaluation software 3.0.2 was used to calculate the KD (BIAcore). Briefly, the slope at 20-s after injection was recorded and the concentration of free MBL in each sample was extrapolated from the calibration curve.

The KD was calculated using the solution affinity model with general fit parameters according to the equation: where Bfree = concentration of free MBL in solution, B = the total concentration of MBL, and A = the total concentration of Glupep ( 23 ). Cell surface ELISA C3 deposition was measured on hypoxic/reoxygenated HUVECs as previously described ( 6 , 18 ). Briefly, confluent monolayers of HUVECs were subjected to either 0, 12, or 24 h of <1% O2 (hypoxia) in a humidified chamber (Coy Laboratory Products, Ann Arbor, MI) at 37°C. Medium was removed and cells were reoxygenated for 3 h in a 95% air/5% CO2 incubator in the presence of 30% human serum diluted in gelatin/Veronal-buffered saline supplemented with 5 mmol/L Ca2+/Mg2+ (GVB2+). Glupep or GlcNAc (Sigma) was diluted to various concentrations in 30% human serum/GVB2+.

Cells were fixed in 1% paraformaldehyde (Sigma) for 30 min and then washed. C3 was detected using a peroxidase-conjugated polyclonal goat anti-human C3 Ab (Cappel, West Chester, PA) and developed with ABTS. VCAM-1 expression was measured using a mAb to human VCAM-1 (mAb 6G10) ( 24 ) as previously described ( 25 ). All ELISA data represent the mean ± SE. All data were analyzed by a two-way ANOVA with pairwise multiple comparisons using the Tukey test. Sigma Stat (Jandel Scientific, San Rafael, CA) was used for statistical analysis. Confocal microscopy Confocal microscopy for MBL deposition was performed as previously described ( 6 , 25 ). Briefly, HUVECs were grown on LabTek tissue culture slides (Nunc, Naperville, IL) and exposed to either 0 or 24 h of hypoxia.

The hypoxic medium was removed and GVB2+ containing 30% human serum treated with either PBS (vehicle), an inhibitory mAb specific for human MBL (3F8, 5 μg/ml), or Glupep (30, 10, or 3 μg/ml) was added at initiation of the 3-h reoxygenation period. Cells were washed and fixed in 4% paraformaldehyde for 15 min and blocked with 10% normal goat serum. Human MBL was identified using a biotinylated 1C10 mAb and streptavidin-conjugated Cy5 (blue; Jackson ImmunoResearch, West Grove, PA). The cells were washed, counterstained with propidium iodide (20 μg/ml), mounted, and analyzed as previously described ( 6 , 25 ). Results To determine whether the peptide SFGSGFGGGY (Glupep) is capable of inhibiting the binding of MBL to a natural ligand, GlcNAc, we performed competition experiments using a BIAcore 3000.

As expected, purified recombinant human MBL bound to immobilized BSA-GlcNAc conjugate (Fig. 1 ⇓ , sensogram A). A functional inhibitory mAb to MBL (3F8, 5 and 50 μg/ml) inhibited this interaction (sensograms C and D, respectively), indicating that the binding of MBL to BSA-GlcNAc was specific. Preincubation of MBL with Glupep inhibited rMBL binding to BSA-GlcNAc in a concentration-dependent manner (Fig. 2 ⇓ ). Glupep also inhibited native human MBL (purified from human serum) in a comparable manner (data not shown). Thus, our data suggest that a short amino acid sequence from cytokeratin 14 is capable of binding MBL and attenuating its interactions with a natural ligand.

Question: Collagen, elastin, keratin, myosin, and lectin are?
Answer: Proteins