Abstract
Dietary proteins undergo luminal digestion by gastric and pancreatic proteases and then membrane digestion by peptidases associated with the brush border membrane of enterocytes. The end products of this digestion are predominantly dipeptides and tripeptides and, to a lesser extent, free amino acids. Peptides are absorbed across the brush-border membrane via a proton-coupled peptide transporter whereas free amino acids are absorbed via multiple amino acid transporters. Once inside the cell, dipeptides and tripeptides are hydrolyzed into free amino acids by cytosolic peptidases. Amino acids then exit across the basolateral membrane via specific amino acid transporters. There are several genetic diseases related to amino acid transport defects in the intestine; clinical consequences of these defects are dependent on specific amino acids whose absorption is impaired in each of these defects. To date, there are no genetic diseases identified in peptide transport in the intestine that result in significant clinical consequences.
Keywords
Protein digestion, Proteases and peptidases, Dipeptides and tripeptides, Proton-peptide cotransport, Amino acid transport, Hartnup disease, Cystinuria, Lysinuric protein intolerance, Microbial ecology
47.1
An Overview of Protein Digestion and Absorption
Amino acids constitute an important class of nutrients obligatory for normal function and survival of mammalian cells. These amino acids are classified as essential and nonessential purely based on whether or not the cells have the ability to synthesize them. If the cells have the capacity to generate certain amino acids endogenously using other nutrients or metabolites as precursors, such amino acids are called “nonessential”; the amino acids that cannot be synthesized by mammalian cells are called “essential.” But in biological sense, all amino acids that are necessary for synthesis of cellular proteins are essential. If any of the proteinogenic amino acids, whether “essential” or “nonessential,” is missing, cells cannot synthesize proteins. Nutritional needs of amino acids in human and in other animals are met by assimilation of dietary proteins in the small intestine. The process involves digestion of proteins in the intestinal lumen to generate products of smaller size that are absorbable by the enterocyte. Interestingly, the end products of protein digestion in the intestinal lumen are not exclusively free amino acids, but a mixture of free amino acids and small peptides. The intestinal epithelium has efficient transport mechanisms to absorb from the lumen not only free amino acids but also dipeptides and tripeptides. The absorbed small peptides are however digested into free amino acids in the cytoplasm of the enterocytes, which then exit the cell to enter the portal circulation. This characteristic is unique to protein assimilation because in the case of carbohydrates, digestion in the intestinal lumen must be completed to yield monosaccharides before absorption into the enterocyte can occur.
An additional feature that is unique to protein assimilation is the need for the presence of multiple transport systems in the intestinal epithelium to handle the end products from digestion. Proteins are made up of 20 different amino acids that are distinct from each other in terms of their chemical structure, which determines their size, lipophilicity, and electrical charge. Because of their widely different physicochemical properties, a single transport system cannot handle all amino acids. It is even more complicated in the case of peptides, where the possible number of chemically distinct forms is 400 for dipeptides and 8000 for tripeptides. Interestingly, intestinal absorptive cells express multiple transport systems to absorb the 20 different amino acids from the lumen but only a single transport system for the 400 different dipeptides and 8000 different tripeptides. As the enterocyte is polarized and absorption of the digestion products from the lumen into blood across the intestinal epithelium is a vectorial process, it requires transport systems in the brush border membrane for entry into the cell and transport systems in the basolateral membrane for exit out of the cell. These two membranes express different sets of transport systems to facilitate vectorial transfer of protein digestion products. Furthermore, the basolateral membrane has to express transport systems, which will enable the cells to obtain amino acids from blood for cellular function during intervals between meals.
A scheme of intestinal assimilation of proteins is shown in Fig. 47.1 . A typical western diet contains ~ 100 g protein/day. In addition to the proteins in the diet, salivary and gastrointestinal secretions contain a significant amount of protein that is digested and absorbed in the gastrointestinal tract. Furthermore, intestinal cells are constantly sloughed off into the lumen due to normal turnover of the epithelial cell layer; proteins in these sloughed cells are also digested and absorbed for reuse of the constituent amino acids. Luminal digestion of these exogenous and endogenous proteins is carried out by gastric and pancreatic proteases. The resultant end products, mostly large peptides, undergo further hydrolysis by a variety of peptidases present in the brush border membrane of the intestinal epithelium. Analysis of luminal contents after a protein meal has shown that amino acids are present in the lumen primarily in peptide form rather than in free form. The peptides in the lumen consist mostly of 2–6 amino acids, and the concentrations of peptide-bound amino acids are as high as 80% of total amino acids. Free amino acids are absorbed into enterocytes across the brush border membrane via multiple amino acid transport systems. Small peptides consisting of two or three amino acids are transported intact across the brush border membrane via a specific peptide transport system. Thus, the protein digestion products enter the enterocyte primarily in the form of dipeptides and tripeptides. Transport of free amino acids contributes relatively less to the entry of protein digestion products into the enterocyte. Nonetheless, the protein digestion products enter portal circulation mostly as free amino acids because of the efficient intracellular hydrolysis of peptides by cytoplasmic peptidases. Peptides resistant to cytosolic peptidases may be transported intact across the basolateral membrane, but the contribution of this route to the total absorption of protein digestion products is minimal.
47.2
Role of Gastric and Pancreatic Proteases in Protein Digestion
Digestion of proteins in stomach involves the protease pepsin, which is secreted by the chief cells in stomach as the inactive precursor pepsinogen. This zymogen is activated by acidic pH in the stomach lumen. High levels of H + cause a conformational change in pepsinogen and expose the catalytically active site. The resultant active form of pepsinogen then acts on inactive pepsinogen and generates pepsin by limited proteolysis. Pepsin then acts on inactive pepsinogen to generate more pepsin by autocatalysis. Pepsin is an acid protease optimally active under acidic conditions. This is facilitated by the involvement of two aspartic acid residues at the active site of the enzyme. Since the two aspartic acid residues need to function alternatively as acids and bases in the catalytic process associated with protein digestion, the enzyme works optimally at a pH that is close to the pKa of the carboxylic acid group of these aspartic acid residues (~ pH 3). The end products of pepsin action on proteins are large polypeptides. The role of pepsin in protein digestion is limited because the enzyme is active only in the stomach and loses its activity when it enters the small intestine where the luminal pH is neutral.
When the stomach contents enter the small intestine, endocrine cells in the duodenum and jejunum are exposed to an acidic pH, which stimulates the secretion of the hormone secretin from specific enteroendocrine cells (S cells). This hormone acts on the acinar cells of the exocrine pancreas and the cholangiocytes lining the bile ductules to induce secretion of bicarbonate. Bicarbonate-rich pancreatic and biliary secretions reach the duodenum where they serve to neutralize rapidly the acid. Polypeptides and fats in the stomach contents also act on enteroendocrine cells in the duodenum and jejunum (I cells) to induce the secretion of another hormone, cholecystokinin. This hormone induces the secretion of pancreatic fluid rich in digestive enzymes. Cholecystokinin also causes contraction of the gallbladder and at the same time relaxes the sphincter of Oddi to release bile into the duodenum. Thus, the entry of stomach contents into small intestine stimulates the delivery of bicarbonate, bile, and pancreatic digestive enzymes to the intestine. Unlike pepsin, pancreatic digestive enzymes are optimally active at neutral pH. Therefore, neutralization of the acidic pH by bicarbonate in small intestine is critical for the activity of these enzymes.
Pancreatic secretions contain several enzymes relevant to protein digestion, but all of them are secreted as inactive precursors. These are trypsinogen, chymotrypsinogen, proelastase, and procarboxypeptidases. The first step in the activation of these zymogens is the activation of trypsinogen, which takes place in the lumen of the small intestine. Enteropeptidase associated with the brush border membrane of the intestinal epithelial cells is responsible for this process, which involves limited proteolysis of trypsinogen to generate the active enzyme trypsin. Trypsin then acts on chymotrypsinogen, proelastase, and procarboxypeptidases and generates the active forms of these enzymes: chymotrypsin, elastase, and carboxypeptidases. These pancreatic proteases then act on the polypeptides entering the small intestine from stomach and generate smaller peptides consisting of 6–8 amino acids. Free amino acids are also produced to a small extent by the action of the carboxypeptidases. Trypsin acts on peptide bonds formed by the carboxyl group of cationic amino acids (arginine and lysine). Chymotrypsin prefers to hydrolyze peptide bonds formed by the carboxyl group of aromatic amino acids (phenylalanine, tyrosine, and tryptophan). Elastase hydrolyzes peptide bonds formed by the carboxyl group of aliphatic amino acids (glycine, alanine, valine, leucine, and isoleucine). The differential specificity of these proteases for peptide bonds in polypeptides makes the digestive process very efficient. The end products of protein digestion by pancreatic proteases consist predominantly of peptides with 6–8 amino acids. Free amino acids comprise only a small fraction of the products of protein digestion by pancreatic proteases.
47.3
Role of Membrane-Bound and Cytoplasmic Peptidases in the Enterocyte in Protein Digestion
The peptides arising from protein digestion by pancreatic proteases are subjected to further hydrolysis by peptidases associated with the brush border membrane of enterocytes. The active sites of these peptidases are located on the luminal side of the membrane (i.e., these peptidases are ectoenzymes) and hence the resultant products are released into the intestinal lumen. The specificity of these peptidases is toward oligopeptides consisting of 6–8 amino acids. The final end products are predominantly small peptides containing 2–3 amino acids. These dipeptides and tripeptides are transported into enterocytes via a specific transport system in the brush border membrane. Once inside the cells, the small peptides are broken down by cytoplasmic peptidases to release free amino acids. The basolateral membrane of the enterocyte possesses a number of amino acid transport systems that are responsible for the exit of amino acids from the cell into portal circulation. Peptides that escape hydrolysis by cytoplasmic peptidases enter portal circulation via a peptide transport system present in the basolateral membrane that is distinct from the brush border membrane peptide transport system, but this constitutes a relatively minor component of the absorptive process. Protein digestion products enter portal circulation predominantly in the form of free amino acids.
47.4
Sites of Absorption of Protein Digestion Products
Stomach plays a negligible role in the absorption of protein digestion products. The small intestine is the principal site of protein absorption. By the time the luminal contents reach the ileocecal junction, absorption of proteins is almost complete. The colonic epithelium does possess an appreciable capacity to absorb protein digestion products, but its physiological significance in the overall process of absorption of dietary proteins is questionable. It is possible that bacterial proteins are digested and absorbed to a significant extent in the colon.
Within the small intestine, there are regional variations in the absorptive capacity for protein digestion products. The two groups of end products, namely, amino acids and di- and tripeptides, are absorbed at different rates in different sections of the small intestine. The absorptive capacity for dipeptides and tripeptides is greater in proximal small intestine than in distal small intestine, whereas in the case of amino acids the absorptive capacity is greater in the distal small intestine than in the proximal small intestine. These differential gradients in the transport capacities for amino acids and small peptides along the jejuno-ileal axis may have physiological relevance. Digestion of proteins in the intestinal lumen by pancreatic proteases releases primarily large peptides, which are not absorbable as such. It is the action of the membrane-bound peptidases in the brush border membrane of enterocytes that generates a major portion of the absorbable products, namely, amino acids and di- and tripeptides. Although these peptidases are present throughout the small intestine, their activities are much higher in ileum than in jejunum, implying that the ileal brush border membrane is capable of more extensive hydrolysis of peptides than the jejunal brush border membrane. It is therefore conceivable that as the luminal contents move along the intestine from jejunum to ileum, the rate of appearance of free amino acids in the lumen gradually increases, while the luminal concentration of di- and tripeptides gradually decreases. The parallelism between the absorptive capacities for amino acids and di- and tripeptides and the luminal concentrations of the corresponding substrates along the jejuno-ileal axis enhances the efficiency of the absorptive process.
Numerous studies have demonstrated that the colonic epithelial cells are also capable of absorption of amino acids as well as peptides. Even though only very small amounts of proteins and protein digestion products enter the colon under normal physiological circumstances, it is conceivable that the large intestine serves a useful function in special situations such as in the immediate postnatal period or in patients with ileostomies. Furthermore, the large intestine contains appreciable quantities of bacterial proteins and their degradation products. Amino acids and di- and tripeptides arising from these bacterial proteins may be absorbed in the colon. Bacteria also generate certain d -amino acids that can be absorbed via specific amino acid transporters expressed predominantly in the large intestine. Alternatively, the peptide transport system in the colon might have a totally different function unrelated to amino acid nutrition. Bacteria generate chemotactic peptides such as muramyl dipeptide and N -formyl-Met-Leu-Phe; these might be the physiological substrates for the peptide transport system in the colon to modulate host-bacteria communication.
47.5
Generation of Driving Forces for Amino Acid and Peptide Absorption in the Enterocyte
Absorption of amino acids and peptides across the intestinal epithelium is mediated by multiple transport systems that are expressed differentially in the lumen-facing brush border membrane and the blood-facing basolateral membrane. The transport processes that occur via these systems fall into two categories: active and passive. Active transport processes are energized by some form of driving force and are able to mediate uphill movement of their substrates against an electrochemical gradient. In contrast, passive transport processes do not depend on any form of driving force and consequently mediate the movement of their substrates only down an electrochemical gradient.
The driving force for active transport in the intestinal brush border and basolateral membranes comes from transmembrane ion gradients and membrane potential. Fig. 47.2 details the mechanisms responsible for the generation of these driving forces. The ultimate energy source for these processes is ATP. Na + -K + ATPase, located exclusively in the basolateral membrane of enterocytes, uses ATP to mediate the uphill transport of Na + from the cell into blood and uphill of transport of K + from blood into the cell. This generates an inwardly directed Na + gradient (∆ pNa) and an outwardly directed K + gradient (∆ pK) across the basolateral membrane. Since the Na + :K + stoichiometry for this process is 3:2, the transport system also generates an inside-negative membrane potential (∆ ψ). The K + channel located in the basolateral membrane mediates the efflux of K + down its concentration gradient, providing an additional mechanism for the generation of the inside-negative membrane potential. The brush border membrane expresses NHE3, an isoform of Na + -H + exchanger, which uses the transmembrane Na + gradient as the driving force to facilitate the efflux of H + from the cell into intestinal lumen. This generates a transmembrane H + gradient (∆ pH) across the brush border membrane. This active efflux of H + is responsible for the acidic microclimate pH that exists on the luminal surface of the brush border membrane. This creates approximately a 10-fold concentration gradient for H + (outside > inside) across this membrane. There is also a Cl − channel in the brush border membrane which mediates the efflux of Cl − into intestinal lumen. The luminal fluid contains substantial amounts of Na + and Cl − arising from dietary sources and salivary and gastrointestinal secretions. Thus, there are five different driving forces, namely, an inwardly directed Na + gradient, an inwardly directed H + gradient, an inwardly directed Cl − gradient, an outwardly directed K + gradient, and an inside-negative membrane potential, which provide energy to support the active transport processes mediated by various amino acid and peptide transport systems in the brush border and basolateral membranes.
47.6
Entry of Protein Digestion Products Into Enterocytes Across the Brush Border Membrane
47.6.1
Amino Acid Transport
In the past, there have been several reviews classifying the amino acid transport systems present in the intestinal brush border membrane. In recent years, specific proteins responsible for these amino acid transport systems have been cloned and characterized. Table 47.1 classifies these transport systems and lists their substrate specificity and dependence on ion gradients. Most of these transport systems are active and mediate uphill transport of their substrates. This characteristic is important for the transport systems in the intestinal brush border membrane because the physiological function of these transport systems is to effectively absorb amino acids from the intestinal lumen. If this process is mediated by energy-independent facilitative transport systems, the absorptive process will not be complete, resulting in significant loss of amino acids in the feces. Fig. 47.3 describes how each of the amino acid transport systems in the intestinal brush border membrane is coupled to its driving forces, indicating the directionality of the movement of the amino acid substrates and the cotransported ions.
| Transport System | Molecular Identity | HUGO Nomenclature | Substrates | Ion Dependence |
|---|---|---|---|---|
| B 0 | B 0 AT1-ACE2 (heterodimer) | SLC6A19-ACE2 | Neutral amino acids | Na + |
| B 0,+ | ATB 0,+ | SLC6A14 | Neutral amino acids Cationic amino acids | Na + , Cl − |
| X − AG | EAAC1/EAAT3 | SLC1A1 | Anionic amino acids | Na + , K + , H + |
| ASC | ASCT2 | SLC1A5 | Neutral amino acids (Ala, Ser, Cys, Gln, Asn) | Na + |
| b 0,+ | b 0,+ AT-rBAT (heterodimer) | SLC7A9-SLC3A1 | Neutral amino acids Cationic amino acids Cystine | None |
| IMINO | IMINO-ACE2 (heterodimer) | SLC6A20-ACE2 | Imino acids | Na + , Cl − |
| N | SNAT5 | SLC38A5 | Neutral amino acids | Na + , H + |
| PAT | PAT1 | SLC36A1 | Small neutral amino acids | H + |
| β | TauT | SLC6A6 | β-Amino acids (taurine, β-alanine) | Na + , Cl − |
47.6.1.1
System B 0
The major transport system responsible for the transport of neutral amino acids across the intestinal brush border membrane was originally identified as the system B, now as system B 0 , to highlight its broad (“B” for broad) substrate specificity with selectivity for neutral amino acids (superscript “0” indicating amino acids with no net charge). This transport system is Na + -dependent and accepts all neutral amino acids that possess the amino group in the α-position as substrates. Imino acids and β-amino acids, though neutral in terms of electrical charge, are excluded by the system. Cationic and anionic amino acids are also not substrates for this transport system. Since the transport function of system B 0 involves symport of Na + and neutral amino acids, the transport process is electrogenic. Therefore, under physiological conditions, an inwardly directed Na + gradient and an inside-negative membrane potential provide driving force for this system. The molecular identity of the protein responsible for this transport system has been established recently. The protein is identified as B 0 AT1, meaning “the first transport protein responsible for the amino acid transport system B 0 .” The human B 0 AT1 consists of 634 amino acids. According to the Human Genome Organization (HUGO) nomenclature, this transporter belongs to the solute-linked carrier (SLC) gene family SLC6 and is identified as SLC6A19. The gene coding for this protein is located on human chromosome 5p15.33.
B 0 AT1 is expressed in the apical membrane of small intestinal and renal epithelial cells. Interestingly, the recruitment of this transporter to the apical membrane depends on two different associated proteins: one specific for the small intestine and the other for the kidney. An ectopeptidase, known as angiotensin-converting enzyme 2 (ACE2), is associated with intestinal B 0 AT1. ACE2 is a carboxypeptidase and is located in the intestinal brush border membrane. This protein is obligatory for proper recruitment of B 0 AT1 to the brush border membrane. In the absence of ACE2, B 0 AT1 is not recruited to the brush border membrane. The enzyme activity of ACE2 also plays an essential role in the activity of B 0 AT1. ACE2 hydrolyzes neutral amino acids from the carboxy terminus of the peptides present in the intestinal lumen and the released amino acids serve as the substrates for B 0 AT1. A related protein, known as collectrin, serves a similar role for the renal B 0 AT1.
47.6.1.2
System B 0,+
System B 0,+ is similar to system B 0 but accepts neutral amino acids as well as cationic amino acids ; this unique substrate specificity is indicated by the superscript “0,+.” This transport system is energized not only by a transmembrane Na + gradient but also by a transmembrane Cl − gradient, and the transport process is electrogenic. Thus, there are three driving forces for this transport system: an Na + gradient, a Cl − gradient, and the membrane potential. The protein responsible for the activity of system B 0,+ has been cloned. The common name for the transporter is ATB 0,+ (amino acid transporter B 0,+ ). It belongs to the transporter gene family SLC6 and is identified as SLC6A14. The transport characteristics of the cloned protein are similar to those described for system B 0,+ . The expression of ATB 0,+ is more predominant in large intestine than in small intestine. A unique feature of ATB 0,+ is its ability to transport several amino acids in their d -isomeric form. Since colonic bacteria produce d -amino acids, ATB 0,+ expressed in the brush border membrane of colonocytes might play a role in the absorption of these amino acids. Even though it is generally believed that d -amino acids do not participate in mammalian metabolism, it is becoming increasingly evident in recent years that this may not be true. For instance, d -serine has recently been identified as the endogenous activator of N -methyl- d -aspartate receptor in glutamatergic neurons. Therefore, the ability of ATB 0,+ to transport d -amino acids may have physiological significance. The gene coding for the human protein is located on chromosome Xq23-q24.
The unusually broad substrate selectivity of this transport system highlights the potential of this transport system for the delivery of amino acid-based drugs and prodrugs. ATB 0,+ transports various nitric oxide synthase inhibitors and the antiviral agents valacyclovir and valganciclovir.
47.6.1.3
System b 0,+
A high-affinity, Na + -independent transport system for neutral and cationic amino acids is present in the intestinal brush border membrane. The lack of Na + dependence is the primary characteristic that distinguishes system b 0,+ from system B 0,+ . Interestingly, system b 0,+ is also capable of transporting the disulfide amino acid cystine (Cys-S-S-Cys). This is the primary transport system for the absorption of cystine in intestine and kidney. System b 0,+ functions as a heterodimer, consisting of two different proteins. The heavy subunit of this transport system is known as rBAT (i.e., related to b 0,+ amino acid transport) which in itself has neither any transport function nor membrane topology characteristic of an authentic transporter. The light chain, known as b 0,+ AT (i.e., b 0,+ amino acid transporter) is responsible for the transport function. rBAT heterodimerizes with b 0,+ AT during biogenesis via disulfide cross-linking and facilitates the trafficking of the heterodimer to brush border membrane. In this respect, the function of rBAT in the trafficking of b 0,+ AT is similar to that of ACE2 in the trafficking of B 0 AT1. Another interesting feature of this transport system is that it functions as an obligatory amino acid exchanger. Under physiological conditions, it mediates the entry of cationic amino acids and cystine into enterocytes in exchange for neutral amino acids. Thus, the absorption of cationic amino acids and cystine via this transport system is coupled to the release of neutral amino acids into intestinal lumen. As the entry of cationic amino acids into the cell is coupled to the efflux of neutral amino acids, the transport process becomes electrogenic. Therefore, the inside-negative membrane potential provides the driving force for the entry of cationic amino acids. rBAT, also known as SLC3A1, belongs to the SLC3 gene family and the gene coding for this protein is located on human chromosome 2p16.3-p21. b 0,+ AT, also known as SLC7A9, belongs to the SLC7 family of amino acid transporters and the gene coding for this protein is located on human chromosome 19q13.1.
47.6.1.4
IMINO System
The IMINO system of the intestinal brush border membrane is exclusive for imino acids such as proline, hydroxyproline, and pipecolic acid. It is present in jejunum as well as ileum. The transport process is Na + -dependent. In addition to Na + , Cl − also plays an obligatory role in the catalytic process. The stoichiometry for Na + :Cl − :proline is 2:1:1, rendering the transport process electrogenic. The human transporter is identified as SLC6A20. It is also referred to as SIT1 (i.e., sodium-coupled imino acid transporter 1). The gene coding for the transporter is located on human chromosome 3p21.3.
47.6.1.5
System β
Among the amino acid transport systems in the intestinal brush border membrane, system β occupies a unique position because, unlike other transport systems, it recognizes taurine, a nonprotein amino acid, as a high-affinity substrate. Studies with purified intestinal brush border membrane vesicles have shown that this transport system interacts exclusively with β-amino acids. System β has no affinity for α-amino acids. Anionic and cationic amino acids are also excluded by this system. Among the β-amino acids, taurine shows the highest affinity. This transport system has an absolute requirement for Na + as well as Cl − , being energized by transmembrane gradients for Na + and Cl − . The Na + :Cl − :taurine stoichiometry is 2 or 3:1:1, which makes the transport process electrogenic. This transport system is sensitive to inactivation by Ca 2 +. The protein responsible for the transport function of system β has been identified at the molecular level. This transporter is known as TAUT (taurine transporter) and identified as SLC6A6. The gene coding for the transporter protein is located on human chromosome 3p26-p24.
47.6.1.6
System X − AG
System X − AG is defined as the transport system that transports the anionic amino acids aspartate and glutamate exclusively and with high affinity. There is evidence for the existence of this transport system in small intestine. System X − ag shows an absolute dependence on Na + , being energized by an inwardly directed Na + gradient. The presence of an outwardly directed K + gradient markedly stimulates the Na + -dependent activity of this system, implying that the movement of Na + and the amino acid from outside to inside is coupled to the movement of K + from inside to outside. The transport process is electrogenic, resulting in the transfer of a positive charge across the membrane. Since aspartate and glutamate exist as monovalent anions under physiological conditions, the electrogenic nature of the transport system suggests that multiple Na + ions are involved in the catalytic process. The simplest stoichiometry of Na + :amino acid:K + is 3:1:1. The activity of system X − AG is also modulated by H + , and now it appears that H + is indeed an additional cotransported ion. At the molecular level, the protein responsible for the transport activity of system X − AG is known as EAAT3 (excitatory amino acid transporter 3) or SLC1A1. The gene coding for the transporter is located on human chromosome 9q24.
47.6.1.7
System ASC
The intestinal brush border membrane also possesses a transport system selective for small neutral amino acids such as alanine, serine, and cysteine; based on this substrate specificity, the system is known as ASC (i.e., alanine, serine, cysteine). The protein responsible for the transport function has been cloned. The cloned transporter is an Na + -dependent obligatory amino acid exchanger. The transport function involves the entry of Na + and a neutral amino acid into the cell coupled to the efflux of Na + and a neutral amino acid out of the cell. The transporter is now referred to as ASCT2, the second member of the ASC amino acid transporter family. ASCT2 (SLC1A5) belongs to the SLC1 gene family and the gene coding for the protein is located on human chromosome 19q13.3.
47.6.1.8
System N
System N, an Na + -coupled transport system that is specific for glutamine, asparagine, and histidine, is expressed in the brush border membrane of intestinal crypt cells. Two different proteins have been identified at the molecular level, which mediate glutamine transport with characteristics similar to those of system N; these are named SN1 (also known as SLC38A3) and SN2 (SLC38A5). Of these, SN2 is predominantly responsible for the uptake of glutamine across the brush border membrane of intestinal crypt cells. SN2 has been cloned ; it transports its amino acid substrates in an Na + -coupled manner in exchange for intracellular H + . Therefore, the Na + /glutamine entry into crypt cells via SN2 is coupled to the efflux of H + . Consequently, the transport activity of SN2 is associated with intracellular alkalinization. This feature may be of biological importance in crypt cells. Since these cells proliferate at a faster rate, they need glutamine as an obligatory nitrogen source for de novo production of purine and pyrimidine bases necessary for DNA/RNA synthesis. In addition, the intracellular alkalinization associated with SN2 transport activity serves to promote cell proliferation. Therefore, SN2 in crypt cells may be a critical determinant of intestinal cell proliferation. The gene coding for SN2 is located on human chromosome Xp11.23.
47.6.1.9
System PAT
A proton-coupled electrogenic transport system for short-chain amino acids (glycine, alanine, and proline) has been described in the brush border membrane of the human colon carcinoma cell line Caco-2. The protein responsible for this activity is called PAT1 (proton-coupled amino acid transporter 1). It is identified as SLC36A1 and the gene coding for the transporter is located on human chromosome 5q33.1. This protein is expressed exclusively in the intestinal brush border membrane in humans and mediates the H + -coupled electrogenic transport of amino acids such as glycine, alanine, and proline. PAT1 also transports taurine and the photodynamic therapy agent 5-aminolevulenic acid.
47.6.2
Peptide Transport
47.6.2.1
Independence of Peptide Transport From Amino Acid Transport
Amino acids and peptides are absorbed in the small intestine by different mechanisms. The most convincing evidence for the presence of a distinct transport mechanism for small peptides is that in the amino acid transport defects Hartnup disease and cystinuria, the affected amino acids are poorly absorbed when presented in free form, but the absorption is normal when these amino acids are presented as small peptides. Furthermore, there is no competition between amino acids and small peptides for intestinal absorption and also when compared on the basis of equivalent amino acid composition, small peptides are absorbed more efficiently than free amino acids. Maximal capacity to absorb free amino acids versus small peptides also differs along the longitudinal axis of the small intestine. These findings clearly indicate that amino acid absorption and peptide absorption are two totally different processes.
47.6.2.2
Driving Force for Peptide Transport
Transport of intact small peptides across the intestinal brush border membrane is an active process. The dependence of the process on metabolic energy is indicated by the demonstration of inhibition of peptide transport by hypoxia, ATP depletion, and metabolic inhibitors such as cyanide and dinitrophenol. The dependence of the transport process on Na + is at best partial in intact cells, whereas the transport process in brush border membrane vesicles is totally Na + -independent but still electrogenic. Because the charge transfer occurs even in the cases of peptides that predominantly exist as zwitterions under experimental conditions, it is apparent that peptides are cotransported with an ion other than Na + . A clue to the identification of this ion first came from studies in which peptide transport was found to be stimulated by an inwardly directed H + gradient. The H + dependence of intestinal peptide transport has now been widely accepted. The acidic microclimate pH that normally exists on the luminal surface of the intestinal brush border membrane provides the driving force for the peptide transport system ( Fig. 47.4 ). The mechanism for the generation of the chemical gradient for H + across the brush border membrane is the activity of NHE3, an isoform of the Na + -H + exchanger.
47.6.2.3
Influence of Chain Length on Peptide Transport
Most studies on peptide transport have employed only dipeptides as substrates. There is, however, considerable evidence for the acceptance of tripeptides by the intestinal peptide transport system. Transport of tetrapeptides occurs only to a small extent, if at all. Peptides with chain length greater than four amino acids do not appear to be absorbed via mediated pathways in the intestine. There is ample evidence for significant absorption of longer peptides, but the process may involve non-mediated mechanisms.
47.6.2.4
Molecular Identity of the Intestinal Peptide Transport System
The protein responsible for the intestinal peptide transport activity has been cloned. The cloned transporter, known as PEPT1 (peptide transporter 1), mediates H + -coupled electrogenic transport of dipeptides and tripeptides. This transporter, designated SLC15A1, belongs to the SLC15 gene family and the gene coding for the protein is located on human chromosome 13q33-q34. The human PEPT1 consists of 708 amino acids and immunolocalization studies have shown unequivocally the expression of this transporter in the intestinal brush border membrane. The minimal structural features required for recognition by PEPT1 consist of the charged amino and carboxyl groups separated by a carbon backbone with a distance of 5.5–6.3 Å. This essential structural requirement accommodates dipeptides and tripeptides but not free amino acids and peptides longer than tripeptides. Even though there are 400 different dipeptides and 8000 different tripeptides potentially present in the intestinal lumen as the result of digestion of dietary proteins, PEPT1 is solely responsible for handling this wide array of peptides. The affinity of PEPT1 for its peptide substrates is low, with the Michaelis constant in the submillimolar range. This makes sense in terms of physiological function of this transporter. The concentration of dipeptides and tripeptides resulting from the digestion of proteins in the intestinal lumen can be as high as 100 mM. Therefore, the presence of a low-affinity and high-capacity transport system is highly suitable for the absorption of these peptides under these conditions. The broad substrate selectivity of this transport system represents a unique feature that has received increasing attention because of the potential of such a system for oral delivery of drugs and prodrugs. PEPT1 does indeed possess the ability to transport a variety of drugs. This includes β-lactam antibiotics, angiotensin converting enzyme inhibitors, and anticancer drugs, among others. PEPT1 also transports p -aminophenylacetic acid, ω-amino fatty acids, 5-aminolevulinic acid, valacyclovir, and valganciclovir.
PEPT1 is expressed throughout the small intestine and the capacity of this transporter for the absorption of dipeptides and tripeptides is very high. Under normal conditions, very little protein digestion end products leave the terminal small intestine. PEPT1 is also expressed, though at lower levels, in the large intestine. The expression of the transporter in colon is induced under certain pathological conditions. Colonic expression of PEPT1 has been noted in patients suffering from short bowel syndrome and inflammatory bowel disease. In short bowel syndrome, significant quantities of protein digestion end products may pass through the shortened small intestine unabsorbed and enter the large intestine. The expression of PEPT1 in colon may serve to absorb the peptides as a compensatory mechanism. Similarly, in inflammatory bowel disease, the expression of PEPT1 in colon may be related to the ability of the transporter to transport the bacteria-derived chemotactic peptide N -formyl-Met-Leu-Phe and pro-inflammatory peptides l -Ala-γ- d -Glu-meso-DAP and muramyl dipeptide. Transport of these peptides via PEPT1 might facilitate the communication between the host and the colonic bacteria involving the host immune system. Immune cells present in the lamina propria also express PEPT1 ; these cells are likely exposed to these immune modulators via PEPT1 after transfer across the brush border membrane.
47.7
Fate of Absorbed Amino Acids and Peptides in the Enterocyte
The peptide transport system localized in the brush border membrane of enterocytes transports dipeptides and tripeptides into the cell, followed by intracellular hydrolysis of the transported peptides ( Fig. 47.1 ). Thus, intracellular peptidases in enterocytes are vital in the terminal stages of protein assimilation. Enterocytes are rich in peptidase activity against small peptides, and these enzymes act primarily on dipeptides and, to a lesser extent, on tripeptides. The specificity of these peptidases with respect to the amino acid chain length of the peptide substrates fits well with the corresponding specificity of the peptide transport system in the brush border membrane. In addition to hydrolyzing the exogenous peptides arising from absorption from the lumen, the intracellular peptidases in enterocytes also participate in the breakdown of endogenous proteins. Accordingly, the activity of these enzymes increases during starvation, a condition associated with enhanced protein breakdown in enterocytes. In contrast, if animals are put on a protein deficient but isocaloric diet, a condition that leads to a decrease in the entry of exogenous peptides into enterocytes, the activity of these enzymes decreases.
It is likely that some of the absorbed peptides resistant to hydrolysis by intracellular peptidases appear intact on the serosal side. This is especially true in the case of peptides containing proline and hydroxyproline that are known to be relatively resistant to hydrolysis by cellular peptidases. Gardner et al. have reported that approximately 10% of the amino nitrogen entering the mesenteric blood during absorption of a partial hydrolysate of casein in vivo is in the form of peptides. Obviously, the extent of amino nitrogen absorption in the form of intact peptides across the enterocyte is influenced by the amino acid composition and the amino acid sequence of the dietary proteins because these factors determine the susceptibility of the peptides arising from luminal digestion to hydrolysis by brush border and cytosolic peptidases in enterocytes.
Free amino acids arising either from hydrolysis of peptides within enterocytes or from transport from the lumen into enterocytes feed into several metabolic pathways, namely, degradation, conversion into other amino acids, incorporation into proteins, and transport into blood. The small intestine is a metabolically active tissue. It is a site of synthesis of mucins and apolipoproteins, and its cell renewal rate is very rapid. These metabolic processes utilize amino acids that enter into the cell either from the lumen or from blood. Glutamine, glutamate, and aspartate are quantitatively the most important amino acids as respiratory fuel in the intestinal epithelium. The metabolic products of these amino acids that appear in the intestinal venous blood include CO 2 , NH 3 , lactate, citrulline, and alanine. The capacity of the intestinal epithelium to utilize glutamine, glutamate, and aspartate is underscored by the observations that very little glutamate and aspartate enter the blood even when the animals are fed a high concentration of these amino acids. Arginine is also extensively metabolized in the small intestine. A significant fraction of the absorbed amino acids is utilized in protein synthesis in the small intestine.
47.8
Exit of Protein Digestion End Products Across the Basolateral Membrane
47.8.1
Amino Acid Transport
There are at least six amino acid transport systems in this membrane, two of them being Na + -dependent (system A and system GLY), three being Na + -independent (y + , L, and T), and one being Na + -independent or Na + -dependent depending on the substrate (y + L) ( Table 47.2 ; Fig. 47.5 ). The Na + -independent pathways are responsible for the transport of amino acids from the cell into blood, thus participating in the overall process of transcellular absorption of amino acids from the intestinal lumen, whereas the Na + -dependent pathways play a role in supplying the intestinal absorptive cells with amino acids for cellular nutrition during periods between meals.
| Transport System | Molecular Identity | HUGO Nomenclature | Substrates | Ion Dependence |
|---|---|---|---|---|
| y + L | y + LAT1-4F2hc (Heterodimer) | SLC7A7-SLC3A2 | Neutral amino acids Cationic amino acids | Na + None |
| A | ATA2/SNAT2 | SLC38A2 | Neutral amino acids | Na + |
| GLY | GLYT1 | SLC6A9 | Glycine | Na + , Cl − |
| y + | CAT1 | SLC7A1 | Cationic amino acids | None |
| L | LAT2-4F2hc (heterodimer) | SLC7A8-SLC3A2 | Neutral amino acids | None |
| T | TAT1 | SLC16A10 | Aromatic amino acids | None |
47.8.1.1
System A
System A is Na + -dependent and accepts all neutral amino acids including imino acids as substrates. Ghishan et al. have shown that one of the transport pathways for glutamine in the intestinal basolateral membrane is Na + -dependent. This pathway, from its substrate specificity, appears most likely to be system A. This is an electrogenic transport system and derives its energy from an Na + -gradient as well as the membrane potential. The orientation of these driving forces in vivo is appropriate for active transport of amino acids from blood into intestinal cells. Therefore, this system does not play any role in the exit of protein digestion end products from enterocytes into portal circulation. Its physiological role is to mediate the entry of amino acids such as glutamine into enterocytes from blood. This might be of importance to maintain the amino acid nutrition of the intestinal cells when entry of amino acids across the brush border membrane is limited as may be the case between meals or during illness. Three different proteins, namely, ATA1-3 (amino acid transporter A 1-3), which exhibit transport characteristics similar to those of system A, have been cloned. Tissue expression pattern of these transporter proteins suggests that ATA2 (also known as SNAT2 for sodium-coupled neutral amino acid transporter 2) is most likely the transporter responsible for system A activity expressed in the intestinal basolateral membrane. SNAT2 is expressed ubiquitously in mammalian tissues, including the small intestine. It mediates Na + -dependent electrogenic transport of short-chain neutral amino acids. Glutamine is an excellent substrate for this transporter, a finding relevant to glutamine uptake by enterocytes from blood. SNAT2 is expressed exclusively in the basolateral membrane of the intestinal epithelium. SNAT2 (SLC38A2) belongs to the SLC38 gene family and the gene coding for the transporter is located on human chromosome 12q.
47.8.1.2
GLYT1 (Glycine Transporter 1)
Glycine is a constituent of glutathione, an antioxidant tripeptide found in high concentrations in intestinal epithelial cells. The availability of glycine controls the levels of glutathione in enterocytes. There are two different transporters for glycine in mammalian cells, namely, GLYT1 and GLYT2. Of these, GLYT1 is expressed in the intestine. Immunohistochemical studies have established the preferential localization of the transporter in the basolateral membrane of enterocytes. The transport of glycine via GLYT1 is coupled to the movement of Na + and Cl − , with an Na + :Cl − :glycine stoichiometry of 2:1:1. Thus, the transport process is electrogenic. The role of GLYT1 in vivo is to import glycine into enterocytes from blood for cellular utilization in metabolic pathways such as glutathione synthesis. But when intracellular levels of glycine are high enough, the transporter might also be able to mediate the efflux of this amino acid from the cells into blood.
47.8.1.3
System y +
System y + is defined as the system that transports cationic amino acids (lysine, arginine, and ornithine) by an Na + -independent mechanism. It is present in the intestinal basolateral membrane. It is unlikely that this system plays any role in the exit of cationic amino acids from enterocytes into portal circulation because of the inside-negative membrane potential in these cells. As is the case with system A, system y + may function to maintain the amino acid nutrition of intestinal cells during times of limited amino acid entry across the brush border membrane. Three different proteins, known as CAT1-3 (cationic amino acid transporter 1-3), have been cloned which exhibit transport features similar those of system y +. Among these, CAT1 is responsible for system y + in the basolateral membrane of enterocytes. This transporter, identified as SLC7A1, belongs to the SLC7 gene family and the gene coding for the protein is located on human chromosome 13q12-q14.
47.8.1.4
System L
System L is the major Na + -independent system in the intestinal basolateral membrane for the transport of neutral amino acids. Imino acids, though zwitterionic in nature, are excluded by the system. Recent studies on the molecular aspects of this transport system have identified two different isoforms of system L (LAT1 or SLC7A5 and LAT2 or SLC7A8) belonging to the SLC7 transporter gene family. Both of them require 4F2hc (heavy chain associated with the 4F2 antigen), also known as CD98, as a common subunit. The 4F2hc-LAT1 and 4F2hc-LAT2 heterodimers behave as obligatory amino acid exchangers. 4F2hc-LAT2 heterodimer is the primary contributor to the activity of system L in the intestinal basolateral membrane. Since this transporter is an obligatory amino acid exchanger, its function will not result in a net influx or efflux of amino acids across the membrane. LAT2 is the actual transporter; 4F2hc plays only an accessory role in bringing LAT2 to the membrane. 4F2hc (SLC3A2) has structural similarity to rBAT and both proteins belong to the same gene family. The gene coding for 4F2hc is located on human chromosome 11q13 and the gene coding for LAT2 is located on human chromosome 14q11.2.
47.8.1.5
System y + L
Transport of cationic amino acids across the basolateral membrane from the cell into blood during absorption has to occur against an electrical gradient, and this is made possible via a separate transport system that couples Na + -dependent entry of neutral amino acids into cells and Na + -independent exit of cationic amino acids. The substrate selectivity for neutral amino acids resembles that of system L and the interaction with cationic amino acids is similar to system y + . These characteristics led to naming this transport system y + L. System y + L exists in two isoforms, each functioning as a heterodimer. As is the case with system L, 4F2hc is the common heavy subunit for both isoforms of y + L. The light subunit, which is the actual transporter, is specific for each isoform. The light subunits are known as y + LAT1 and y + LAT2. Both isoforms are expressed in the basolateral membrane of intestinal epithelial cells. Both y + LAT1 (SLC7A7) and y + LAT2 (SLC7A6) belong to the SLC7 gene family. The gene coding for y + LAT1 is located on human chromosome 14q11.2 and the gene coding for y + LAT2 is located on human chromosome 16q22.1-q22.2.
47.8.1.6
System T
System T is an Na + -independent transporter selective for aromatic amino acids (tryptophan, phenylalanine, and tyrosine). It is expressed in the basolateral membrane of enterocytes. The protein responsible for the transport function has been cloned ; it is known as SLC16A10 or TAT1 (T amino acid transporter 1). It is a uniporter that mediates the efflux of aromatic amino acids from enterocytes into blood. Despite its uniport activity, recent studies with Slc16a10-knockout mice have shown a coupling between this transporter and LAT2 at functional level in which the aromatic amino acids that are effluxed via TAT1 are used by LAT2 for exchange with intracellular neutral amino acids.
47.8.2
Peptide Transport
Dyer et al. was the first to investigate peptide transport in the basolateral membrane. This study has provided evidence for the presence of a peptide transport system in this membrane. The transport system is relatively specific for small peptides. Even though the substrate selectivity of the basolateral membrane peptide transport system is similar to that of the brush border membrane peptide transport system, it is not mediated by PEPT1. Thus, the exit of hydrolysis-resistant dipeptides and tripeptides from enterocytes into portal circulation occurs via a separate peptide transporter ( Fig. 47.4 ). There have been attempts to characterize the basolateral membrane peptide transporter at the molecular level, but these have not been successful in establishing the molecular identity of the protein responsible for this transport process.
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