Biochemistry #01 Bone Formation Dr. Nabil Bashir Farah Banyhany
Greetings This lecture is quite detailed, but I promise you will make it through, it just requires your 100% FOCUS! Let s begin. Today s lecture is about bone metabolism. The following objectives are the highlights of what you should remember by the end of this lecture. The doctor recommended that we go over them one by one, because the exam questions will be restricted to these objectives. Let s begin talking about the biochemical structure of the bone: Learning objectives Describe the biochemical structure of bone composition: inorganic, collagen and non-collagen proteins List bone matrix proteins and functions Review calcium and phosphate homeostasis in relation to PTH, calcitonin and vitamin D Know the biochemical functions of osteoblast and osteoclast bone cells Understand the role of alkaline phosphatase, calcium, phosphate, PTH, calcitonin and vitamin D in bio-mineralization List Bone biomarkers and their clinical significance Bone Composition Inorganic (67%): Hydroxyapatite 3 Ca10(PO4)6(OH)2 Organic (33%) component is called osteoid Type I collagen (28%) Non-collagen structural proteins (5%) 2
What is written above represents the bone composition, which has two major components: inorganic and organic. The inorganic component makes up 2/3 of the bone s composition and is mainly hydroxyapatite, which is made of calcium, phosphorus, and hydroxyl groups. The organic component makes up 1/3 of the bone s composition, and is known as the osteoid. It consists of type 1 collagen (28%) and non-collagen proteins (5%). What is collagen? Collagen is the most abundant protein in the human body and is the substance that holds the whole body together. It is found in the bones, muscles, skin, tendons, connective tissue, nails, and hair where it forms a scaffold to provide strength and structure.it is a fibrous protein (for support) that is composed ofthree polypeptide strands held together in a helical conformation. Apatite Apatite is a group of phosphate minerals and calcium. Hydroxyapatite, Ca10(PO4)6(OH)2: major component of tooth enamel and bone minerals. Fluorapatite, Ca10(PO4)6(F)2 Chlorapatite, Ca10(PO4)6(Cl)2 Bromapatite, Ca10(PO4)6(Br)2 Fluorapatite (or fluoroapatite) is more resistant to acid attack than is hydroxyapatite. For this reason, toothpaste typically contains a source of fluoride anions (e.g. sodium fluoride, sodium monofluorophosphate). Hydroxyapatite represents the major component of the inorganic part of the bone. Keep in mind that hydroxyapatite consists of calcium, phosphorus, and hydroxyl ions. Later on, we will discuss the calcium 3
and phosphorus homeostasis and how their concentrations will affect the bio-mineralization of the bone. As shown on the previous page, there are different types of apatite: flourapatite, chlorapatite, and bromapatite. Flourapatite has a unique and specific quality, in that it is resistant to acid attack compared to hydroxyapatite. This explains why fluoride is added to toothpaste, because it will resist the acid attack on the teeth. ------------------------------------------------------------------------------------ Collagen There are many types of collagen, in fact more than 20 types exist. Our main concern for this lecture is collagen type I. The difference between one collagen type and another is in the polypeptide chain that composes it. As we know, the synthesis of collagen starts with three polypeptide chains, which could all be the same OR 2 could be the same and 1 different chain. So, according to these combinations, there is a variety of combinations of collagen. Remember that the collagen that composes the organic part of bone constituent is collagen type I. The polypeptide chains in collagen type I are the alpha 1 and alpha 2.It specifically is composed of TWO alpha 1 and ONE alpha 2.The difference 4
between these polypeptide chains is in the amino acid sequence. Each polypeptide chain comes from a gene. Refer to the picture on the previous page: A Brief Explanation on the Synthesis of Collagen: This picture shows the synthesis of collagen, till it turns into a fiber.the primary structure of collagen is quite interesting; it is composed of triple repeats (amino acid sequences or tri-peptides that are repeated many times). These triple repeats are in the following order: glycine, proline, and any other amino acid. The sequence then continues in the same order. These triplet repeats are not seen in globular proteins, hence are only seen in fibrous proteins like collagen. Each polypeptide chain is composed of 1000 amino acids. Next, the three polypeptide chains will intertwine with each other forming a structure called procollagen. Portions of the polypeptide in the N and C terminal are known as signal/signature peptides. These are important in the diagnosis of diseases, which will be discussed in detail later on. After this stage of synthesis, procollagen will be converted to tropocollagen. The difference between tropocollagen and procollagen is that the end regions are removed by specific enzymes (peptidases) in the lumen of the endoplasmic reticulum. Many tropocollagen are then arranged with each other in a staggeredand overlapped fashion to form the collagen fibril. Collagen fibril is a combination of many tropocollagen arranged in a specific way (staggered method).lastly, the combination of many collagen fibrils will form a collagen fiber. This is how collagen fibers/molecules are formed. Due to the large amount of intertwining of the polypeptide chains, you can infer how strong a collagen molecule is, hence its structural function. Collagen type I will go through these stages of synthesis and bones will have many fibers of this type of collagen. 5
-------------------------------------------------------------------------------------------- In further detail, proline will be hydroxylated and lysine will be hydroxylated and oxidized. The hydroxylation of proline and lysine aims in helping the molecules cross-link covalently with each other. So, the polypeptide chains in tropocollagen and collagen fibrils form covalent bonds between oxidized and hydroxylated lysine, and between hydroxylated lysine and hydroxylated proline. These hydroxylations and oxidations of some amino acids take place enzymatically during the protein biosynthesis of collagen processing. The hydroxylases require vitamin C. So, deficiency of vitamin C could lead to weak, non-functional collagen molecules non-healthy bone structure, leading to alot of diseases, such as scurvy. -------------------------------------------------------------------------------------------- Here are some types of non- collagen proteins that are also found in bones: Let us discuss how our body maintains calcium and phosphorus in physiological concentrations or homeostasis, which is important for bone bio-mineralization. The control of calcium and phosphorus takes place by different mechanisms. Among these mechanisms is the hormonal 6
mechanism. Parathyroid hormone, vitamin D, calcitonin, and estrogen are known to have an effect on calcium and phosphorus. Parathyroid hormone (PTH) affects the concentration of calcium when the blood calcium is low. Actions of PTH: 1. Increases bone resorption of calcium and phosphorus. Calcium will come out of the bone, so its concentration will increase (as well as the phosphorus) in blood. 2. Increase the kidney reabsorption of calcium increase the concentration of calcium in the blood. 3. Prevents the reabsorption of phosphorus to prevent hyperphosphatemia. 4. Activates vitamin D productionfrom the kidney. 7
Actions of Vitamin D: After vitamin D has been activated, it will: 1. Increase the calcium and phosphorus absorption from the intestine 2. Increase calcium resorption from the bone 3. Increase the calcium reabsorption from the kidney 4. Helps in calcium mineralization because now that there is an excess in calcium and phosphorus, vitamin D will help in bone mineralization. Now, there are some hormones that counteract the action of PTH and vitamin D, and examples of them are the calcitonin and estrogen. They will counteract the resorption action of vitamin D and PTH from the bone. So, integration between these hormones will maintain the calcium and phosphorus concentration and will play an important role in bone biomineralization. In conclusion, PTH and vitamin Dwill act on bone, kidney, and intestines. Calcitonin and estrogen will counteract the resorption effect of PTH and vitamin D. 8
.. ~These are some of the cellular components (and their characteristics) of bone. ~Resorption: degradation and removal of calcium and phosphorus from the bone. ~Osteoclast will degrade proteins by protease. ~Mineralization: formation of hydroxyapatite in the presence of calcium and phosphorus and the deposition of the hydroxyapatite in the holes that are formed between the collagen fibrils and fibers. ~Osteoblasts activate osteoclasts by specific cell products called RANKL (ligand), a protein that will bind to a receptor on the osteoclasts called RANK receptor. Once RANKL binds to RANK on osteoclasts, osteoclasts will be activated. It will be functional and will start bone resorption. ~Bone formation and bone resorption both are involved in bone remodeling. 9
Mineralization process of bones: There are modulators/effectors that will affect bone mineralization. These modulators could be stimulators/promoters or inhibitors. The promoters are of two types in bone mineralization: An enzyme called tissue nonspecific alkaline phosphatase (TNAP)and phosphatase orphan 1. These are produced within the cells of osteoblasts to help in bone mineralization. The inhibitors consist of inorganic pyrophosphate (PPi, a strong inhibitor of bio-mineralization). When some kinases phosphorylate proteins using ATP, they will take the inorganic phosphate on the ATP and put it on the protein and the rest of the phosphate in the ATP is what is known as the as PPi. This is commonly seen in glycogen metabolism. The other inhibitor is the non-collagenous proteins in the matrix such as osteopontin. 10
The promoters and inhibitors are local modulators: meaning that they are produced from the bone cells and act IN the bone cells for mineralization. The last group of modulators is nonlocal, and it acts as a hormone in a paracrine / autocrine manner. This modulator is called fibroblast growth factor 23. It is synthesized in fibroblasts, it circulates in the blood, and it obviously affects bone mineralization. It will affect different organs (kidney, intestine, and parathyroid) in order to have the calcium and phosphorus for bone mineralization. Pi: inorganic phosphate PPi: inorganic pyrophosphate It is important to remember the ratio above, when discussing bone mineralization. For bone mineralization, we need Pi. When you have high PPi, it will inhibit bone mineralization. So, some modulators (local and nonlocal) will decrease the concentration of PPi and convert it to Pi to stimulate mineralization, controlling its ratio. 11
Key Players in Bone Mineralization: ~Hormones ~Bone cells ~Cell products *They interact with each other to help in bone mineralization. The figure on the next page portrays the process in which calcium and phosphorus are controlled by different modulators. Lets break it down Inside the cell, inorganic phosphate will be accumulated in specific vesicles, as well as the organic phosphate compounds using the phosphatase 1 enzyme that will remove the phosphate from them. The inorganic phosphate and calcium will combine and make the hydroxyapatiteinside the vesicle. The vesicle will move outside to the extracellular matrix (ECM), where more growth of hydroxyapatite and calcification will take place. 12
Pyrophosphate functions as an inhibitor; high concentrations of it will inhibit bio-mineralization. By this, we can infer that a low ratio of Pi/PPi also leads to inhibition. Enzymes and other modulators will try to get rid of PPi or hydrolyze it. An important example is an enzyme called tissue nonspecific alkaline phosphatase (TNAP). This is where alkaline phosphatase plays an important role in biomineralization. TNAP will help in stimulating some enzymes to hydrolyze PPi into Pi. The problem here is that PPi will inhibit this tissue nonspecific alkaline phosphatase as well as inorganic phosphate. Also, the PPi will stimulate the synthesis of a protein (OPN). This protein will inhibit the growth of hydroxyapatite. So, TNAP will dephosphorylate this protein and make it non-active to prevent its function (inhibition of growth and propagation of hydroxyapatite). 13
One of the sources of PPi is metabolism and other dephosphorylation of proteins in the bone cells. Generally speaking, this picture shows the maintenance of high concentration phosphorus. Removing more PPi will help in mineralization by the help of TNAP.The function of TNAP is to dephosphorylate some important proteins that prevent growth and propagation of hydroxyapatite and it will stimulate enzymes that will hydrolyze PPi into Pi. ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ In summary of the picture above, the fibroblast growth factor 23 affects the phosphate concentration in the blood. How? Fibroblast growth factor 23 has many effects such as the prevention of phosphorus reabsorption from the kidney and prevention of the production of PTH. A high amount of phosphate in the blood will affect this growth factor.the take-home lesson about this concept: fibroblast growth factor 23 will try (by certain mechanisms) to decrease the phosphate 14
in the serum to maintain the ratio of Pi/PPi as required by the bone. If the bone needs mineralization, it will increase Pi and if not, it will decrease Pi. 16 Biomarkers that help in the diagnosis of bone diseases: One of the biomarkers required for bone formation is the alkaline phosphatase. Alkaline phosphatase dephosphorylates proteins that prevent mineralization. It also decreases (by an indirect pathway)the PPi, increasing the bone formation, because PPi is an inhibitor for bone formation as mentioned earlier. When looking at the alkaline phosphatase activity, one can see that there is bone formation taking place in that patient. Other markers for bone formation are procollagen type I propeptides propeptides on N and C terminal region are called procollagen I N-terminal peptide (PINP) and procollagen I C-terminal peptide (PICP). 15
If the concentration of procollagen type I propeptides in the blood or urine is high, this means that there is bone formation. The following are markers for bone resorption: Hydroxyproline: if you finda high concentration of the hydroxyproline in a blood or urine sample, then this is an indicator of bone resorption or bone damage. Why is hydroxyproline categorized as a marker for bone resorption? Because it comes from collagen. Collagen is composed of glycine, proline, and other amino acid (triplet repeats). These prolines are hydroxylated. Once you have bone resorption, osteoclasts will degrade proteins, including collagen. As a result of collagen degradation, there will be high amounts of hydroxyproline. Cross linked telopeptides of type 1 collagen (NTX, CTX): There are regions in the N-terminal and C-terminal that make covalent cross links between oxidized lysine and hydroxylysine. When collagen is degraded, these cross links form. High concentration of these cross links (different from the N-terminal of procollagen, which is found in mature collagen) is seen in a degraded mature collagen. Best of luck Edited By: Abdul-Rahim Shilbayeh 16