2.4. Collagen glycosyltransferases
Grassman and his coworkers proved in 1957 that neutral sugars are covalently linked to collagen. Butler, Cunningham, and many others in the late 60’s and early 70’s demonstrated the covalent linkage of the sugars to hydroxylysine, and the carbohydrate units turned out to be either a monosaccharide galactose or a disaccharide glucosylgalactose. These are the only sugars found in the triple helical region of collagens (Myllylä 1976, Kivirikko & Myllylä 1979, Kielty et al. 1993, Prockop & Kivirikko 1995, Bateman et al. 1996). These hydroxylysyl glycosides have also been identified in the C1q subcomponent of complement that contains collagen-like amino acid sequences (Kivirikko & Myllylä 1979). The structure of 2-O-α-D-glycopyranosyl-O-β -D-galactopyranosylhydroxylysine involving an unusual α1→2-O-glycosidic linkage between glucose and galactose is shown in Figure 3.
Figure 3. The structure of the collagen-specific carbohydrate Glc-Gal-Hyl in peptide linkage. The carbohydrate unit may also consist only of the Gal portion (Modified from Kivirikko & Myllylä 1979 with permission of Academic Press).
The carbohydrate units are transferred to collagen as posttranslational modifications catalyzed by two specific enzymes; hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT), transfers galactose to hydroxylysyl residues, and then galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT), transfers glucose to galactosylhydroxylysyl residues. Both enzymes have been demonstrated to exist in a number of tissues (Kivirikko & Myllylä 1979). The extent of glycosylation varies between collagen types and within the same collagen type in different tissues and at different ages (Risteli 1977, Anttinen et al. 1977b, Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982, Kielty et al. 1993).
2.4.1. Purification and molecular properties of collagen glycosyltransferases
2.4.1.1. Hydroxylysyl galactosyltransferase (GT)
Partial purification of this enzyme has been achieved from guinea-pig skin, rat kidney cortex, and human platelets (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982). From chick embryo extract, a method, consisting of six conventional protein purification steps yielded about a 50- to 150-fold increase in specific activity (Risteli et al. 1976a). GT has not yet been isolated as a homogeneous protein, the highest degree of purification, up to about 1000-fold, having been obtained from chick embryo extract by using a procedure consisting of ammonium sulfate fractionation, affinity chromatography on collagen-agarose, and gel filtration (Risteli et al. 1976b). The major problem in the enzyme purification is its marked tendency to be inactivated during purification and in many cases the specific activity decreases during steps in which the purity of the enzyme protein clearly increases (Risteli et al. 1976a, Risteli 1978).
The molecular weight of GT is not known as no homogeneous protein has been isolated and the gene or cDNA for the enzyme has not yet been cloned. The activity of the partially purified enzyme is found by gel filtration to fall into two major peaks with molecular weights of about 450 and 200 kDa, and one minor species with a molecular weight of about 50 kDa (Risteli et al. 1976a). It is not known whether they represent an aggregate of the enzyme alone or the enzyme with some other proteins.
GT is a glycoprotein as its activity can be inhibited by concanavalin A, and the inhibition can be reduced by methyl α-D-mannoside, methyl α-D-glucoside, mannose, fructose, and glucose (Risteli 1978, Kivirikko & Myllylä 1979). The enzyme binds to concanavalin A coupled to agarose and can be eluted with a buffer containing methyl glucoside and ethylene glycol (Risteli 1978).
2.4.1.2. Galactosylhydroxylysyl glucosyltransferase (GGT)
Partial purification of GGT has been achieved from guinea-pig skin, rat kidney cortex, chick embryo cartilage, bovine arterial tissue, human fetal tissues, plasma and platelets (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982). Purifications of over 2000-fold from whole chick embryos and about 160-fold from chick cartilage have also been reported using six conventional protein purification steps (Myllylä et al. 1976). GGT has been isolated as a homogeneous protein from chick embryos by two affinity column methods. The first procedure includes ammonium sulfate fractionation, collagen-agarose and UDPglucose-derivative-agarose affinity chromatographies, and two gel filtrations (Myllylä et al. 1977). The second one consists of ammonium sulfate fractionation, concanavalin A-agarose, collagen-agarose, and UDPglucose-derivative-agarose affinity chromatographies, and one gel filtration (Anttinen et al. 1978).
The molecular weight of GGT from chick embryos is about 72 – 78 kDa seen in SDS-PAGE, and it may consist of only one polypeptide chain (Myllylä et al. 1977). Gel filtration gives a lower and variable molecular weight, probably due to partially adsorption of the molecule to the columns (Anttinen et al. 1977a, Myllylä et al. 1977).
Amino acid analysis indicates that the chick GGT is rich in Glu, Gln, Asp, Asn, Gly, and Ala (Anttinen et al. 1978). It is a glycoprotein having a high affinity for concanavalin A-agarose, and this affinity can be reduced by methyl α-D-mannoside (Anttinen et al. 1977a). The chick GGT is capable of generating strong hydrophobic interaction, probably being part of the explanations for adsorption to gel filtration columns and a marked tendency to form aggregates when concentrated (Anttinen et al. 1977a). Antibody against the pure chick embryo GGT inhibits the transferase activity from different tissues, and gives a single precipitation line of identity with the enzyme from many chick embryo tissues in immunodiffusion (Myllylä 1981). However, the gene or cDNA of GGT has not been cloned so far.
2.4.2. Catalytic properties of collagen glycosyltransferases
GT catalyzes the synthesis of galactosylhydroxylysine by transferring galactose from UDP-galactose to hydroxylysyl residues in peptide linkages. GGT catalyzes the formation of glucosylgalactosylhydroxylysine by transferring the glucose from UDP-glucose to galactosylhydroxylysyl residues (see Figure 4). Both enzymes require a bivalent cation, preferably Mn2+ (Kivirikko & Myllylä 1979).
The free ε-amino group of the hydroxylysyl residue and a nonhelical polypeptide conformation are the absolute requirements for GT and GGT, as the N-acetylation or deamination of the free ε-amino group completely inhibits both reactions, and the triple helical conformation of the substrate prevents the interactions with both transferases (Kivirikko & Myllylä 1984, Kielty et al. 1993). The glycosylation reactions are also affected by the amino acid sequence of the peptide, and the peptide chain length (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1984). The number of -X-Hyl-Gly- sequence in a polypeptide chain play a key role in the overall glycosylation (Anttinen & Hulkko 1980) whereas the amino acid sequences adjacent to the hydroxylysyl residue may be a minor factor. Longer peptides are more effective substrates for the transferases (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1984).
The specificity of GT and GGT for their glycosyl acceptors is very high. GT only attaches galactose to hydroxylysyl residues in peptide linkage, and does not attach a second galactose to galactosylhydroxylysyl residues or a galactose to glucosylgalactosylhydroxylysyl residues. Free hydroxylysine does not act as the sugar acceptor. However free hydroxylysine in high concentration and free galactosylhydroxylysine can inhibit the glycosylation reactions (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982). GGT does not attach glucose to a number of carbohydrates, glycopeptides or glycoproteins, in which the galactose is linked to other sugars or to amino acids other than hydroxylysine. This enzyme also fails to attach glucose to hydroxylysyl residues or glucosylgalactosylhydroxylysyl residues. Galactosylsphingosine is one exception in that it can act in vitro as a good glucosyl acceptor for GGT, probably due to its structural similarity to galactosylhydroxylysine (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982).
Figure 4. Glycosylation reactions catalyzed by peptidyl GT and GGT (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).
The preferential glycosyl donor for GT or GGT is the corresponding UDP glycoside. The Km of UDP-galactose for GT is about 20-30 µM whereas the Km of UDP-glucose for GGT is about 5-30 µM depending on the source from which the enzyme was isolated. The reaction product, UDP, and several other nucleotides (UTP, UMP, ATP, ADP, AMP, CDP etc.) act as inhibitors of both enzymes (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1984).
The pH optimum for both glycosyltransferases is about 7-7.4. The optimal Mn2+ concentration is 0.2-2 mM for partially purified GGT, and this enzyme binds at least two Mn2+ having the Kd values of 3-5 µM (site I) and 50-70 µM (site II) (Myllylä et al. 1979). The Kd value for Mn2+ at site II is probably much higher than the physiological concentration. It is not known whether this site has any significance in vivo (Kivirikko & Myllylä 1984). Fe2+(Kd 5-7 µM) and Co2+ (Kd 30 µM) are the only other metals that can activate GGT at low concentration. They can, however, inhibit the enzyme when the concentration is high (Myllylä et al. 1979). Several bivalent cations are inhibitors of the Mn2+-activated hydroxylysyl glycosyltransferase (Kivirikko & Myllylä 1982). There are no corresponding data available for GT.
The mechanism (shown in figure 5) of the GGT reaction has been investigated in detail with the enzyme purified from chick embryos by analyzing the initial velocity and inhibition kinetics (Myllylä 1976). The data agree with an ordered binding of Mn2+, UDP-glucose, and collagen to GGT, and an ordered release of the glycosylated collagen, UDP, and Mn2+, in which Mn2+ does not need to leave the enzyme between the catalytic cycles (Myllylä 1976). Some kinetic studies have also been carried out on GGT from human platelets (Smith et al. 1977). The data are consistent with the scheme described for chick embryo GGT. No detailed kinetic data is available for GT (Kivirikko & Myllylä 1979, Kivirikko & Myllylä 1982, Kivirikko & Myllylä 1984).
Figure 5. Schematic representation of the mechanism proposed for the GGT reaction. At high Mn2+ concentrations, GGT probably binds two Mn2+ ions successively before binding to UDP-glucose. Due to the high Kd value at site II, this situation may not occur in vivo and therefore is not shown. Mn2+ need not to leave the enzyme between the catalytic cycles. GGT: galactosylhydroxylysyl glucosyltransferase; UDPglc: UDP-glucose; Glc-pept: glucosylated peptide (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).
2.4.3. Intracellular sites of collagen glycosylations
It was proposed earlier based on work using isolated Hela cell membrane fractions that the glycosyltransferases were located only in the plasma membrane, and that the glycosylations occurred immediately before secretion of procollagen from the cells (Hagopian et al. 1968, Bosmann 1969). The assays used, however, were nonspecific, and more evidence accumulated later that suggested the glycosylations take place in a different intracellular location (Kivirikko & Myllylä 1979). The activities of GT and GGT in most cases were found to be associated with the rough ER (Kivirikko & Myllylä 1979), but it was also found to be present in the smooth ER (Harwood et al. 1975b). It has been shown by Blumenkrantz and his coworkers that the distribution of both glycosyltransferases within purified chick embryo bone microsomes is similar to that of LH. About 70-80% of their activities are intramembranous with the remainder intracisternal. The common location of the major portion of LH, GT and GGT activities suggested that they might form a multienzyme complex to preferentially modify certain lysyl residues in nascent procollagen chains as they pass across the membrane of the ER (Blumenkrantz et al. 1984). It has been also demonstrated by Bortolato et al. in the early 90’s that the GGT specific to collagen is located in the rough ER, smooth ER, and Golgi apparatus in the chick embryo liver (Bortolato et al. 1990, Bortolato et al. 1991, Bortolato et al. 1992).
Brownell & Veis (1975) and Harwood et al. (1975b) found galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues in nascent polypeptide chains, suggesting that glycosylations are initiated while the polypeptide chains are still under assembly on the ribosomes. Oikarinen et al. (1976a) studied the time course of the glycosylation of hydroxylysyl residues in chick embryo cartilage cells, and demonstrated that there was no lag between the hydroxylation of lysyl residues and the glycosylation of hydroxylysyl residues. They also found that after a 5-minute pulse-label with 14C-lysine, the reactions continue for about 10 minutes during the chase period in the chick embryo tendon cells (Oikarinen et al. 1976b) and about 20 minutes in the cartilage cells (Oikarinen et al. 1976a). The triple helix is formed in both cell types at about the same time points (Harwood et al. 1975a), thus suggesting that the glycosylations continue until triple helix formation of the pro-α chains. This conclusion was also reached in the studies, which showed that if the triple helix formation is inhibited, the glycosylations are prolonged, and if it is accelerated, the extent of modification is less (Oikarinen et al. 1976b, Oikarinen et al. 1977). These results agree with the data on hydroxylysyl galactosylation (Risteli et al. 1976a) and galactosylhydroxylysyl glucosylation (Myllylä et al. 1975) in vitro, as the two enzymes do not catalyze reactions with triple helical substrates. The triple helices of procollagen form before they move from the cisternae of the rough ER to the Golgi apparatus, indicating that the hydroxylysyl glycosylations are completed within the cisternae of rough ER (Kivirikko & Myllylä 1979, see Figure 6).
Figure 6. Schematic representation of the events in collagen biosynthesis that occur within the cisternae of the rough ER. The propeptide is removed during the translocation across the membrane and hydroxylation of lysyl residues and glycosylation of hydroxylysyl residues are initiated while the polypeptide chains are still being assembled on the ribosomes. The reactions continue after the synthesis of complete pro α-chains until the triple helix is formed. LH: lysyl hydroxylase; GT: hydroxylysyl galactosyltransferase; GGT: galactosylhydroxylysyl glucosyltransferase; Gal: galactose; Glu: glucose (Modified from Kivirikko & Myllylä 1984 with permission of Elsevier Science).
2.4.4. Collagen glycosyltransferase activities in physiological, pathological, and experimental conditions
Considerable differences are found in hydroxylysyl glycosyltransferase activities between different tissues, and in the same tissue under different conditions. Both were found to vary with age, being higher in embryonic than in adult tissues (Anttinen et al. 1977b, Kovanen & Suominen 1989). The developmental patterns of both glycosyltransferase activities were also studied in chick embryos (Risteli 1977). GT and GGT increased up to the 16th day and decreased thereafter, the highest activities being found in the leg tendons of the 16-day-old chick embryos, and the activities in cartilage were higher than in skin or skull (Risteli 1977). During cartilage and bone formation induced by demineralized bone matrix in rats, both activities rose dramatically from day one to reach the highest values on day nine and dropped down slowly afterwards (Myllylä et al. 1981). PH-4, GGT and other enzymes in human serum are often regarded as biomarkers of collagen biosynthesis. It has been reported that prolonged or concentric exercise causes an increase in the activity of GGT in human serum. The most likely explanation is that the prolonged heavy exercise results in protein leakage from muscles and probably from the collagen-synthesizing cells of the connective tissue (Takala et al. 1986, Takala et al. 1989, Virtanen et al. 1993).
It has been shown under experimental conditions that GT and GGT activities increase in lung of hamsters with bleomycin-induced pulmonary fibrosis (Bolarin et al. 1984a), in liver of CF1 female mice with hepatic murine Schistosomiasis mansoni (Bolarin et al. 1985), and in rat liver with carbon tetrachloride-induced fibrosis (Bolarin et al. 1987) whereas both activities decrease in transformed human and chick-embryo cells (Myllylä et al. 1981), and in isolated chick-embryo tendon cells after the administration of cortisol acetate to the chick embryo (Oikarinen 1977).
The GGT activity increases in many pathological conditions, for example dermatological disorders (Kuutti-Savolainen 1979, Kuutti-Savolainen & Kero 1979, Oikarinen et al. 1982a, Oikarinen et al. 1982b, Ala-Kokko et al. 1987), human breast cancer (Bolarin 1983a), human primary hepatocellular carcinoma (Bolarin 1983b), acute viral hepatitis, cirrhotic liver (Bolarin et al. 1984b) and experimentally-induced primary liver carcinoma (Bolarin 1991). The level of GGT is higher than normal in acute myocardial infarction and during subsequent collagen scar formation (Anttinen et al. 1981). GGT activity significantly increases in injured porcine intervertebral discs (Kääpä et al. 1994) or degenerated discs (Kääpä et al. 1995). Increased serum or tissue GGT activity has been also found in fibrosing lung diseases (Anttinen et al. 1985, Anttinen et al. 1986, Poole et al. 1985, Poole et al. 1989), various rheumatic diseases (Myllylä et al. 1989), neuromuscular disorders (Myllylä et al. 1982), and even the adult respiratory distress syndrome (Farjanel et al. 1993). The increase of GGT in these diseases might result from the accelerated collagen biosynthesis or collagen metabolism.
Savolainen et al. (1981) have shown that members of a family with dominant epidermolysis bullosa simplex have a deficiency of GGT. The enzyme activity was low in serum, skin tissue, and cultured skin fibroblasts whereas other intracellular enzymes of collagen biosynthesis showed no abnormality. No molecular biological data is available from the patients.