PART 1 Introduction
4.4. TRANSCRIPTION: SENDING THE MESSAGE
4.5.3. Posttranslational Processing
Making the Product Useful
Often the polypeptide formed from the ribosome must undergo further processing before it can become truly useful. First, the newly formed chain must fold into the proper struc- ture; in some cases, several different chains must associate to form a particular enzyme or structural protein. Additionally, chaperonesare an important class of proteins that assist in the proper folding of peptides. There are distinct pathways to assist in folding polypep- tides. The level of chaperones in a cell increases in response to environmental stresses such as high temperature. Misfolded proteins are subject to degradation if they remain soluble. Often misfolded proteins aggregate and form insoluble particles (i.e., inclusion bodies). High levels of expression of foreign proteins through recombinant DNA technol- ogy in E. colioften overwhelm the processing machinery, resulting in inclusion bodies.
The formation of proteins in inclusion bodies greatly complicates any bioprocess, since in vitro methods to unfold and refold the protein product must be employed. Even when a
cell properly folds a protein, additional cellular processing steps must occur to make a useful product.
Many proteins are secreted through a membrane. In many cases, the translocation of the protein across the membrane is done cotranslationally(during translation), while in some cases posttranslationmovement across the membrane occurs. When proteins move across a membrane, they have a signal sequence(about 20 to 25 amino acids). This signal
Figure 4.7. Translation of genetic information from a nucleotide sequence to an amino acid sequence. (With permission, from M. W. Jensen and D. N. Wright, Introduction to Medical Microbiology, Pearson Education, Upper Saddle River, NJ, 1985, p. 66.)
116 How Cells Work Chap. 4
sequence is clipped off during secretion. Such proteins exist in a pre-form and mature form. The pre-form is what is made from the m-RNA, but the actual active form is the ma- ture form. The pre-form is the signal sequence plus the mature form.
In procaryotes secretion of proteins occurs through the cytoplasmic membrane. In E. coliand most gram-negative bacteria the outer membrane blocks release of the secreted protein into the extracellular compartment. In gram-positive cells secreted proteins readily pass the cell wall into the extracellular compartment. Whether a protein product is re- tained in a cell or released has a major impact on bioprocess design.
In eucaryotic cells proteins are released by two pathways. Both involve exocytosis, where transport vesicles fuse with the plasma membrane and release their contents.
Transport vesicles mediate the transport of proteins and other chemicals from the endo- plasmic reticulum (ER) to the Golgi apparatus and from the Golgi apparatus to other membrane-enclosed compartments. Such vesicles bud from a membrane and enclose an aqueous solution with specific proteins, lipids, or other compounds. In the secretory path- way vesicles, carrying proteins bud from the ER, enter the cisface of the Golgi apparatus, exit the Golgi transface, and then fuse with the plasma membrane. Only proteins with a signal sequence are processed in the ER to enter the secretory pathway.
Two pathways exist. One is the constitutive exocytosis pathway,which operates at all times and delivers lipids and proteins to the plasma membrane. The second is the regu- lated exocytosis pathway,which typically is in specialized secretory cells. These cells se- crete proteins or other chemicals only in response to specific chemical signals.
Other modifications to proteins can take place, particularly in higher eucaryotic cells.
These modifications involve the addition of nonamino acid components (for example, sug- ars and lipids) and phosphorylation. Glycosylationrefers to the addition of sugars. These modifications can be quite complex and are important considerations in the choice of host organisms for the production of proteins. A bioprocess engineer must be aware that many proteins are subject to extensive processing after the initial polypeptide chain is made.
A particularly important aspect of posttranslational processing is N-linked glycosy- lation. The glycosylation pattern can serve to target the protein to a particular compart- ment or to control its degradation and removal from the organism. For therapeutic proteins injected into the human body these issues are critical ones. A protein product may be ineffective if the N-linked glycosylation pattern is not humanlike, as the protein may not reach the target tissue or may be cleared (i.e., removed) from the body before it exerts the desired action. Further, undesirable immunogenic responses can occur if a pro- tein has a nonhumanlike pattern. Thus, the glycoform of a protein product is a key issue in bioprocesses to make therapeutic proteins (see Chapter 14).
The process of N-linked glycosylation occurs onlyin eucaryotic cells and involves both the ER and Golgi. Thus, the use of procaryotic cells, such as E. coli, to serve as hosts for expression of human therapeutic proteins is limited to those proteins where N-linked glycosylation is not present or unimportant. However, not all eucaryotic cells produce proteins with humanlike, N-linked glycosylation. For example, yeasts, lower fungi, and insect cells often produce partially processed products. Even mammalian cells (including human cells) will show altered patterns of glycosylation when cultured in bioreactors, and these patterns can shift upon scale-up in bioreactor size.
The process of N-linked glycosylation is depicted in Fig. 4.8. The pattern shown is
“typical,” and many variants are possible. The natural proteins in the human body usually
display a range of glycoforms; a single form is not observed. A simple sequence of three amino acids, of which asparagine must be one, is required for attachment of N-linked sug- ars and amino sugars. The sequence at the attachment site is Asn-Xaa-Ser/Thr, where Xaa is any amino acid and the third amino acid in the sequence must be serine or threonine.
The process of N-linked glycosylation begins in the ER, where a preformed branched
Figure 4.8. Example of a N-linked glycosylation pathway (Glc =glucose, M =man- nose, GlcNAc =N-acetylglucosamine, F =fucose, Gal =galactose, Sial =sialic acid). The oligosaccharide side-chain is bound to an asparagine (Asn) of the protein. The upper arm represents the a-1,6 arm and the lower one the a-1,3 arm. The parentheses refer to an op- tional fucosylation. The GlcNAc-ase step is important in insect cells, but not mammalian cells. (Courtesy of C. Joosten.)
118 How Cells Work Chap. 4
oligosaccharide (the dolichol pyrophosphate-oligosaccharide) with 14 sugars is trans- ferred to the amino group of asparagine.
The 14-sugar residue is first “trimmed” by a set of specific glycosidases. In yeast, oligosaccharide processing often stops in the ER, leading to simple glycoforms(or high mannose or oligomannose forms). The initial trimming takes place in the ER, followed by transfer to the Golgi apparatus where final trimming occurs, followed by addition of vari- ous sugars or aminosugars. These units are added through the action of various glycosyl- transferases using nucleotide-sugar cosubstrates as sugar donors. In insect cells high levels of N-acetylglucosaminidase activity results typically in dead-end structures with a mannose cap. Complex glycoformshave sugar residues (N-acetyl glucosamine, galactose, and/or sialic acid) added to all branches of the oligosaccharide structure. Hybrid glyco- formshave at least one branch modified with one of these sugar residues and one or more with mannose as the terminal residue.