BCM 3360 Week 3: Yolk Proteins and Plasma Proteins in Vertebrates and Invertebrates

Yolk Proteins and Plasma Proteins

Learning Outcomes

  • Articulate synthesis and posttranslational modifications of vitellogenins in vertebrates and invertebrates.
  • Compare and contrast yolk proteins in vertebrates, Insects, crustaceans and other invertebrates.
  • Explain the major differences in plasma proteins in vertebrates.
  • Explain the biological functions of larval haemolymph proteins

Lecture Outline

  • Introduction
  • Vitellogenins and Yolk Proteins of Vertebrates
  • Vitellogenins and Yolk Proteins of Insects
  • Vitellogenins and Yolk Proteins of Crustaceans and Other Invertebrates
  • Plasma proteins in vertebrates
  • General anatomy of an egg

Vitellogenins

  • Vitellogenin is a large multidomain apolipoprotein typically produced in females but also present at lower levels in males.
    • An apolipoprotein is a protein that binds oil-soluble substances e.g. fat-soluble vitamins, cholesterol and fats forming lipoproteins.
  • Vitellogenin serves as the primary egg-yolk precursor protein and provides the resources necessary for embryogenesis.
  • Its primary function is to bind to and subsequently transport maternal lipids, carbohydrates, metals (Mg, Ca, and Zn), and phosphorous to the oocyte where it is taken up into tissue via receptor-mediated endocytosis.
  • It belongs to a superfamily of genes known as large lipid transport proteins.

Structural Representation of Vitellogenin

  • Vitellogenin's transport functions are due to its ability to bind to lipids and numerous ligands, and this arises from its biochemical structure.
  • The protein is cleaved at various sites in different species, and several structural domains are well conserved across taxa.
  • Example:
    • N-terminus β-barrel (N-sheet), which harbors the receptor binding area of the protein.
    • α-helical domain, which contains a lipophilic cavity implicated in binding to various ligands.

Vitellogenesis

  • Vitellogenesis is the process by which yolk accumulates in the cytoplasm of an ovarian oocyte.
  • It is one of the final stages of egg formation, occurring just prior to deposition of the chorian.
  • Vitellogenesis is a sequential process incorporating the following events:
    1. Induction of vitellogenin synthesis and its release into circulation.
    2. Transport of vitellogenin in the bloodstream or haemolymph to the target tissue.
    3. Specific recognition and uptake of vitellogenin by the growing oocytes.
    4. Chemical changes in the oocytes, and incorporation into the yolk structure.
  • Vitellogenins are significantly altered between their synthesis and their incorporation into the yolk structure of the oocyte.

Vitellogenin Gene Family in Vertebrates

  • The vitellogenin gene family is constituted of variable gene numbers encoding for polypeptides that are precursors of yolk proteins and derivatives in oviparous and ovoviviparous vertebrates.
  • Vertebrate vitellogenins are synthesized in the liver and are transported almost unchanged from liver to the oocyte.
  • Synthesis of vitellogenins in vertebrates is a coordinated endocrine stimulation process that involves brain, ovary, and liver.
  • Production is seasonal or cyclic depending on gonadotropins as well as others factors such as nutritional status, seasonal effects (water temperature).
  • Post-translational modifications include; phosphorylation, glycosylation, and lipidation.

Linear Representation of Vitellogenin Protein

  • Signal polypeptide
  • Heavy chain Lipovitellin (LvH) is with its four subdomains: N sheet, α helix, C sheet and A sheet;
    • N sheet contains a receptor binding site responsible for the interaction with oocyte
    • The α helix subdomain, a site binding zinc ions is localized
    • C-sheet facilitates transport of triglycerides and phospholipids
    • A sheet is alanine-rich and is involved in embryo gluconeogenesis
  • Cleavage sites

Phosvitin (Pv)

  • Phosvitin is a serine-rich polypeptide able to bind phosphates whose negative charge attracts multivalent cations as calcium, magnesium, zinc, and iron.
  • The Pv domain also has glycosylation sites for binding carbohydrates that, together with ions, promote the aqueous solubility of vitellogenins.
  • The Light chain Lipovitellin (LvL) domain also contains glycosylation sites and is able to bind lipids.
  • The von Willebrand Factor type D domain (vWFD) is involved in the vitellogenin folding and dimerization through disulfide linkages depending on cysteine residues.

Vitellogenesis in the Ovarian Follicle

  • During the vitellogenesis in the ovarian follicle, the endosomes containing vitellogenins are acidified by the action of proton pumps and the cathepsin D.
  • This triggers their cleavage into; lipovitellins, phosvitins and others (\beta-c, and Ct)
  • Thus, this cleavage into two major types of yolk proteins in vertebrates; lipovitellin (lipovitellogenins) and phosvitins.
  • Yolk proteins are rich in amino acids, phosphate, carbohydrate, lipids, vitamins, and mineral ions.
  • Yolk proteins have non-enzymatic action in these animals and hence can be compared to milk proteins in mammals or the storage proteins of plant seeds.
  • NOTE:
    • The vitellogenin gene family in vertebrates includes a variable number of paralog genes and existence of multiple copies due to whole genome duplication events.

Functions of Vitellogenin Genes

  • Because of the existence of multiple copies of these genes in vertebrates, they perform other functions:
    • Their multivalent pattern recognition receptor selectively binds to conserved components of bacteria and virus aiding in immune functions
    • They also have antioxidant activity protecting against oxidative damage
  • NOTE:
    • The synthesis of vitellogenins can be induced by exposure to endocrine-disrupting chemicals (Bisphenol A, Phthalates, Polychlorinated biphenyl, heavy metals e.g. lead, etc) frequently found in polluted environments.
    • Thus, the levels of vitellogenin in teleosts (fishes) is a key role as a biomarker in assessing the EDC effects

Insects Vitellogenins and Yolk Proteins

  • Insects vitellogenins are synthesized in the fat body
  • The insect fat body is analogous to vertebrate adipose tissue and liver
  • The fat body tissues are major sites for nutrient storage, energy metabolism, innate immunity, and detoxification
  • It also plays a central role in the integration of hormonal and nutritional signals to regulate:
    • larval growth, body size, circadian clock, pupal diapause, longevity, feeding behavior, and courtship behavior, partially by releasing fat body signals to remotely control the brain

Synthesis of Vitellogenins in Insects

  • Synthesis of vitellogenins in the fat body is a process that involves substantial structural modifications including glycosylation, lipidation, phosphorylation, and proteolytic cleavage before its secretion and transport into the haemolymph
  • Only minor changes occur after uptake into the oocytes
  • The extent to which vitellogenins are processed in the fat body vary greatly among different insect species
  • In many insects e.g. Honeybee and Moth, vitellogenins are the predominant haemolymph proteins during vitellogenesis while in others like cockroach, fruit fly, the proportion is only about 1 %.

Regulation of Synthesis in Insects

  • In most insects, synthesis is regulated by the juvenile hormone, secreted by glands near the brain.
  • The hormone affects the process of molting, the periodic shedding of the outer skeleton during development, and in adults it is necessary for normal egg production in females.
  • In addition to juvenile hormone, 20-hydroxyecdysone is also responsible for the control of vitellogenin synthesis.
  • Apart from the egg-laying queens, vitellogenins are also present in freshly hatched queens and in workers but is absent in drones.
  • In the bumble bee (Bombus terrestris), the workers produce vitellogenins only when they lack a queen.

Vitellogenesis: Endocrine Regulations

  • Endocrine regulation of yolk protein synthesis in insects is species dependent guided by their individual life style and adaptations
  • (https://www.youtube.com/watch?v=7oe6wb5rKSk)

Crustaceans Yolk Proteins

  • In crustaceans, the egg yolk contains one or more lipoproteins (lipovitellins)
  • The lipovitellin of the small brine shrimp is colored due to the presence of the carotenoid canthaxanthin
  • Other crustacean lipovitellins have a higher lipid content and 2-11 different apolipoproteins with varied molecular masses
  • Vitellogenin synthesis also occurs in the ovary in other decapods (crabs, lobsters, shrimps) and in fat tissues of amphipods and isopods

Yolk Proteins in Nematodes

  • In the nematode Caenorhabditis elegans, the yolk contains two different lipoprotein complexes, of which the A-complex contains three different polypeptides and the B-complex has only one type
  • In these organisms, vitellogenins are synthesized in the intestine of the adult hermaphrodite and transported into the germline
  • Yolk complexes are secreted into the pseudocoelom (body cavity), from where they pass through the gonadal basal lamina and through the sheath pores of the somatic gonad before uptake by maturing oocytes via receptor-mediated endocytosis
  • Yolk proteins serve as a source of nutrients during embryogenesis

Plasma Proteins

  • Plasma proteins are a diverse group of molecules with various structures and functions in blood plasma.
  • These proteins play important roles including providing colloid-osmotic pressure and acting as buffers.
  • However, their concentration differs depending on species, the developmental stage and the physiological conditions
  • Plasma proteins have specific roles; transport of substances, defense reactions, blood clotting

Plasma Proteins of Vertebrates

  • There are several protein super-families in the blood plasma of mammals classified according to their functions in man, and presumably all other vertebrates.
  • The major plasma proteins of man and other mammals are shown in the table below;
  • With the exception of the albumins, all plasma proteins are glycoproteins.
  • The carbohydrate portion increases the solubility of the plasma proteins due to the presence of many polar groups.

Albumin Family of Proteins

  • Albumin family of proteins consists of:
    • Serum albumin
    • Alpha (α) -fetoprotein (AFP),
    • Vitamin-D binding protein (group-specific component)

Serum Albumin

  • This is an abundant protein in the human plasma and in healthy adults’ its normal range is 3.5-5.5 g/dl and is synthesized in hepatocytes.
  • It is monomeric, multi-domain protein, with low molecular mass (66-69 kDa), thus is the main factor determining the colloid-osmotic pressure of the plasma.

Serum Albumin - Binding Sites

  • Domain III has a high-affinity fatty-acid-binding site
  • The binding site for bilirubin lies on domain II and that for indole on domain I.
  • There is a marked species specificity in the binding properties of the albumins; e.g
    • bilirubin binds much more strongly to human and chicken albumin than to bovine or rabbit albumin;
    • frog albumin, in contrast to the others, cannot bind tryptophan

Serum Albumin - Transport Protein

  • It is a transport protein and of greatest physiological importance, is the binding capacity of albumin for organic anions like gall pigments, steroid and thyroid hormones, fatty acids and drugs.
  • Thus, imparts significant role in the pharmacokinetics and pharmacodynamics of many drugs and other important molecules.
  • In humans, albumin acts as markers for the detection and monitoring of diseases, progression, prognosis, or assess the clinical status of patients.
  • It also forms the largest fraction in the lower vertebrates
  • In some organisms e.g. Rana catesbeiana (American bullfrog), albumin concentration is very low (<1 mglml) and only increases markedly during metamorphosis

α-fetoprotein

  • This is major protein of the embryonic plasma of all mammalian embryos
  • It has a molecular mass of about 68 kDa consists of single polypeptide chain comprised of 590 amino acid residues
  • In humans, the maximal concentration of 3 mg/ml is reached in the 13th week of embryo development while in adults, it is found at only ng/ml concentrations.
  • In contrast to the non-glycosylated albumins, α-fetoprotein is glycosylated and has at least two oligosaccharide chains
  • In mammalian foetus, α-fetoprotein is made in the yolk-sac, liver and gut wall.
  • High levels of α-fetoprotein occur in liver regeneration or liver tumors thus it used as biomarker for liver tumors (https://youtu.be/N8vsrUVvokU)
  • In non-mammalian lineages, larval α-fetoprotein is present reptiles but not in the fish or amphibians

Vitamin D-binding protein

  • Vitamin D-binding protein is a multifunctional protein, secreted by the liver.
  • Its principal role is the transporting of vitamin D and its metabolites in blood
  • Since it has actin-binding affinity, vitamin D binding protein also plays a role in removal of any actin that escapes into the circulation when cells are damaged or destroyed
  • Its circulating concentrations are increased by pregnancy and estrogen therapy and are decreased in conditions associated with hypoproteinemia (e.g., liver disease, malnutrition, nephrotic syndrome) and inflammation in humans.

Transferrin

  • These are glycoproteins found in vertebrates which bind and consequently mediate the transport of iron (Fe) through blood plasma.
  • They are produced in the liver and contain binding sites for two (Fe) ions.
  • Free ferric (Fe^{3+}) ions cannot exceed a concentration of 10^{-17} mol/L in neutral solution without forming insoluble ferric oxide.
  • Vertebrates exhibit a very intensive iron metabolism in connection with haemoglobin synthesis
  • Note:
    • Transferrin family of proteins also include: ovotransferrin in bird eggs and lactotransferrin (lactoferrin) in the milk of all mammals (refer to week 2)

Transferrin Affinity and Activity

  • The affinity of transferrin for iron is high and is conserved in mammalian species.
  • The transferrins also have antimicrobial activity, since they sequester essential iron by virtue of their high affinity inhibiting microbial growth
  • It is a dimeric transmembrane protein with two polypeptides of 90 kDa and three N-bound oligosaccharide chains, phosphoric acid and fatty acid residues.
  • The binding of transferrin to the receptor is not species specific
  • However, the receptors of higher mammals bind transferrin of other placentalia but not that of pouched animals (marsupials), birds or amphibians

Transferrin Gene Conservation

  • The gene is conserved with molecular masses of the serum transferrin polypeptide chains, ranging from 61 to 87 kDa in fish, birds and mammals.
  • However, they show high variability in terms of carbohydrate fraction, i.e. sialic acid content and in partly allelic variation in the amino acid sequence
  • An allele is a variant of a gene where the DNA sequence differs between two or more variants
  • Little is known about iron transport proteins in invertebrates though (Fe^{3+}) transport has been demonstrated some species
  • Haemolymph protein of horseshoe crab (Limulus Polyphemus) and butterfly (Manduca sexta), has iron-binding sites.

Haptoglobin

  • Haptoglobins are glycoproteins which bind in a ratio of 1:1 to the haemoglobin released from disintegrated red blood cells.
  • Haptoglobin-haemoglobin complex is taken up and metabolized by cells of the reticuloendothelial system; the haptoglobin molecules are not recycled
  • They are widely distributed in mammals and birds but have not yet been definitely identified in amphibians
  • The haptoglobin molecule is a tetramer with two types of subunit bound by disulphide bridges
  • Canine haptoglobin lacks the disulphide bridges between the dimers
  • Chicken haptoglobin is very different, both structurally and functionally from that of mammals.

Chicken Haptoglobin

  • The chicken haptoglobin is a single chain alpha-acid glycoprotein.
  • It also binds only iso-specific haemoglobin whereas mammalian hepatoglobin binds all haemoglobins including that of chicken

Haemopexins

  • These are glycoproteins that bind free haem and transport it to the liver cells, where it is degraded to gall pigments and the iron is bound to ferritin; the haemopexin returns to the bloodstream (recycled)
  • It exists in all classes of vertebrates and apparently has a relatively low rate of evolution
  • Similar to haptoglobin, chicken haemopexin from that of mammals in terms of carbohydrate components.

Larval Haemolymph Proteins of Insects

  • Larval haemolymph proteins have high aromatic amino acid content and are synthesized in the fat bodies thus are also referred to as arylphorins.
  • The biological functions of larval hemolymph provide amino acids for the synthesis of structural and nutritional substances during metamorphosis, including the aromatic amino acids for the sclerotization of the cuticle.
  • The larval haemolymph may also function as transport proteins for ecdysteron
  • In terms of composition, they have a very unusual amino acid spectrum with a total of 17-26 % phenylalanine and tyrosine, small amounts of carbohydrates and a lipid component

Distribution of Haemolymph Proteins

  • In terms of distribution:
    • only one type of arylphorin monomer is found in the lepidopterans Calpodes ethlius, Heliothis zea, Hyalophora cecropia and Papilio polyxenes and in the honey bee Apis mellifera
    • two occur in the lepidopterans Bombyx mori, Galleria melonella and Manduca sexta and in the cockroach Blatta orientalis;
    • three occur in the dipterans Drosophila melanogaster, Ceratitis capitate and Musca domestica.
    • Sarcophaga peregrina, Lucilia styga and Calliphora vicina there are several other components in addition to the main subunit
  • They are synthesized only in the final larval stage and reach higher concentrations in females than in males
  • Insects also have methionine-rich proteins, histidine-rich protein and those that contain bound riboflavin and copper in their haemolymph

Plasma Lipoproteins

  • These are transport lipoproteins for hydrophobic lipids to the sites of resorption, de novo synthesis, conversion, and use, hydrophobic lipids.
  • Vertebrates and insects have spherical lipid protein particles with very different molecular architectures.

Plasma Lipoproteins of Vertebrates

  • Lipoproteins of all vertebrates correspond in their basic structure to the human forms, a nucleus of neutral lipids surrounded by a layer of proteins and polar lipids (phospholipids, cholesterol).
  • The specific density is mainly determined by the ratio nucleus: coat.

Examples of Lipoproteins

  • Examples: chylomicrons, very-low-density lipoproteins (VLDL), low-density (LDL) and high-density lipoproteins (HDL)
  • The different types of lipoprotein particles have different transport functions.
  • In most vertebrates, fats reabsorbed in the small intestine are transported away by chylomicrons, which are formed in the cells of the intestine and are introduced into the blood via the lymph.
  • In birds, however, the reabsorbed lipids are included into VLDL and directly enter the blood in the portal vein.
  • The chylomicrons are rapidly degraded in the blood.
  • Lipoprotein lipases, located in particular on the surface of capillary endothelial cells, release fatty acids and monoacylglycerols, which are transported to the neighbouring cells with the help of serum albumin.

Lipoprotein Metabolism

  • The remains of the chylomicrons, which retain only the proteins apoB and apoE and are rich in cholesterol esters, are taken up into liver cells.
  • Fats synthesized in the liver are secreted into the blood as VLDL; the HDL is also formed in the liver.
  • In contrast, the fatty acids released from fat tissues are bound directly to serum albumin.
  • The VLDL particles formed in the liver serve primarily to transport triacylglycerols or fatty acids to the peripheral tissues.
  • Due to the action of lipoprotein lipases, the VLDL particles lose triacylglycerols and are converted to particles of intermediate density (IDL).
  • Non-esterified cholesterol of the VLDL is transferred to HDL, esterified by lecithin: cholesterol acyltransferase and returned to the IDL.

Plasma Lipoproteins of Insects and Other Invertebrates

  • Lipid transport in insects is fundamentally different from that in vertebrates.
  • In invertebrates, the triacylglycerols form the non-polar nucleus of lipoprotein particles.
  • Their fatty acids can be released only by radical reshaping of the particle.
  • In insects, fatty acids are transported as strongly polar diacylglycerols (DAGs) arranged close to the surface of the lipoprotein particles, from whence they can be easily removed.
  • Thus, in contrast to the vertebrates, the lipid transport particles of insects are reusable transport proteins.
  • In the migratory locust Locusta migratoria, the half-lives of apolipoproteins and DAGs is 5-6 days and 2-3 h, respectively.

Lipophorins

  • Diacylglycerols and phospholopids are the predominant lipids; triacylglycerols are only found in trace amounts
  • The lipid transporting proteins of insects are called lipophorins whose lipid content is normally 40- 50 % but varies with the metabolic state.

Blood Clotting

  • In the animal kingdom there are three mechanisms for reducing blood losses from damaged blood vessels:
    1. Contraction of the edges of the wound and the vessels.
    2. Blockage of the wound by aggregates of blood cells.
    3. Clotting of the blood fluid.
  • Blood clotting has been observed in the vertebrates, arthropods and certain molluscs (oysters) and in the coelom fluids of sipunculids, brachiopods, echinoids and holothurians.
  • In the case of the invertebrates, biochemical data are available only for the arthropod groups Xiphosura, Crustacea and Insecta

Blood Clotting in Vertebrates

  • The formation of a solid blood clot in all vertebrates involves the polymerization and precipitation of fibrin; this occurs by partial proteolysis of the fibrinogen in the blood plasma.
  • The protease responsible is thrombin.
  • Most clotting factors are serine proteases
  • The zymogens of the clotting cascade are homologous in their C-terminal region (250 amino acids) with the catalytic domain of the pancreas proteases; their N-terminal sequences.

Fibrinolysis and Clotting Inhibitors

  • Fibrin clots are solubilized by the protease plasmin, which is formed from plasminogen
  • The bovine and porcine plasminogens have 78-83 % similarity with that of man
  • A system of clotting inhibitor factors prevents the clotting of blood in intact vessels.
  • The most important regulator of blood clotting is, the C-protein which inactivates factors Va and VIlla and stimulates fibrinolysis.
  • The activated form of the C-protein, is a disulphide-linked dimer of 420 amino acids made up of a light chain with carboxylglutamic acid residues and a heavy chain with the catalytic centre of a serine protease.

Activation of C-Protein

  • The activation of C-protein is catalyzed by thrombin, especially when this is bound to thrombomodulin, which is an integral membrane protein of the endothelial cells in the vessel walls
  • Activated C-protein requires the S-protein which is a vitamin K-dependent protein.

Blood Clotting in Arthropods

  • The clotting capacity of arthropod blood varies considerably according to the species, the developmental stage and the physiological state.
  • It ranges from the complete absence of plasma clotting to the formation of blood clots whose strength exceeds those of mammals
  • The arthropods demonstrate all possibilities for the origin of coagulogen from blood cells
  • In the crustaceans, only from the haemolymph is involved, whilst in the insects both cellular and extracellular proteins appear to be involved.
  • The clotting system includes the coagulogen and serine proteases.
  • The activated factor C converts factor B into its active form, which in turn promotes the conversion of pro-clotting enzyme to clotting enzyme

Arthropod Blood Clotting Mechanisms

  • The clotting reaction is not a proteolytic process but involves the transglutaminase-catalysed formation of pseudopeptide bonding between glutamate and lysine residues
  • Amongst the insects, there is a wide spectrum of blood clotting phenotypes, but in every case specific types of blood cell (coagulocytes) are involved.
  • In some species an aggregate of coagulocyte is formed and surrounded by a network of granular fibrils
  • In other species single coagulocytes appear to send out thread-like processes
  • and in some cases, there is no indication of any plasma clotting

Clotting in Cockroach and Locust

  • In cockroach and locust, a haemocyte coagulant secreted from the blood cells forms an insoluble clot by interacting with the plasma coagulant already present in the haemolymph.
  • Their plasma coagulant is identical to the lipid-transporting protein lipophorin proteolytic enzyme involved in the activation reaction of the xiphosurans.
  • The clotting enzyme is a serine protease is present in the blood cells as a zymogen.
  • The haemocytes of Limulus and Tachypleus contain an inhibitor of clotting, with significant homology to rabbit a-lactalbumin

Clotting in Crustaceans

  • These can be subdivided into three groups based upon their clotting type;
    • In group A (e.g. Cancer and Maja), there is only cell aggregation;
    • In group B (e.g. Macropipus, Carcinus, Galathea, Homarus) plasma clotting follows cell aggregation;
    • In group C (e.g. Astacus, Panulirus) the solidification of the plasma spreads out from blood cells.
  • These coagulogens appear to be structurally quite similar as they all react with the same clotting enzyme.

Antifreeze Proteins

  • The blood plasma of teleosts in the polar oceans and in the coastal waters, which are cold in winter, of the northern temperate zone contains large quantities of special proteins that depress the freezing point of the plasma by several degrees.
  • There are several types of such antifreeze proteins with the same function but very different structures.
  • These antifreeze polypeptides may be subdivided into three basically different classes:
    • Class I includes alanine-rich polypeptides of 3-5 kDa with a secondary structure of amphiphilic a-helices.
    • Antifreeze proteins of class II have so far been detected in only one species of the Cottidae, the Arctic Sea raven Hemitripterus americanus

Antifreeze Protein Classes II and III

  • These are larger proteins (14 kDa) with 8 % cysteine and they contain many reverse turns and five disulphide bridges.
  • The proteins assigned to class III are of intermediate size (6-7 kDa) and have no particularly characteristic amino acid composition or secondary structure. Class III proteins
  • The variety and distribution of the three AFP classes suggests that they arose independently and relatively recently
  • The biosynthesis of both AFPs takes place in the liver.
  • Antifreeze proteins which give rise to thermal hysteresis have been detected in a series of terrestrial arthropods: beetle species from six different families, a cockroach, a bug, a scorpion fly and even a spider.

Frost Resistance in Insects

  • In many insect species frost resistance is achieved not by the prevention of haemolymph freezing by special proteins or other substances but by the converse mechanism of ice nucleation: specific haemolymph proteins (ice nucleators) lead to rapid ice formation in the extracellular fluid under freezing conditions, thereby preventing lethal intracellular ice formation.