Collagen, hydroxyproline formation

The present work was undertaken in an attempt to shed light on the mechanism of hydroxyproline formation in collagen biosynthesis and on the site of action of ascorbate. It was recognized that these phenomena were very likely to be closely interrelated. Proline labeled with tritium was used because it was anticipated that tritium released from proline during hydroxylation would appear in the tissue water as tritiated water. The use of tritiated proline in such a system might then provide a new parameter for following the hydroxylation reaction, provided that the formation of tritiated water was stoichiometrically related to the formation of hydroxyproline. Such an approach might conceivably permit the demonstration of hydroxylation and peptide bond formation as separate chemical steps. 

A more elaborate and somewhat speculative scheme for the formation of collagen and the conversion of proline to collagen hydroxyproline is ... 

If this scheme is correct, it should be possible to obtain conditions under which protein synthesis but not hydroxylation is blocked. That this may be possible experimentally is suggested by the results of recent experiments (24) in our laboratory, which have shown that in the presence of puromycin, the formation of collagen hydroxyproline determined in carageenin tumors as described here is markedly reduced, but that hydroxylation (as determined by the formation of tritiated water and tritiated hydroxyproline) continues. Further work—especially efforts directed at isolation of intermediates—are in progress. 

Gly-Pro-Y > Gly-X-Pro > Gly-X-Y. In a given polypeptide chain of native tropocollagen, about one third of the molecule contains the Gly-Pro-Hyp sequence and two thirds involve Gly-X-Y, which decreases the stability of the triple helix. Amino acid residues other than proline and hydroxyproline that occupy the X- and Y-positions decrease helix stability but are essential for the next level of organization of collagen—the formation of microfibrils. 

Deprivation of ascorbic add results in the inability to form collagen, as measured by hydroxyproline formation. Ascorbic acid given to scorbutic animals 10-12 days after wounding induced hydroxyproline for-... 

Little is known about the nature of this conversion. Presumably the major amount of hydroxyproline formation occurs when proline is bound in peptide linkage in collagen. This was suggested first by Stetten and Schoenheimer (133). The work of numerous subsequent investigators have supported this (133-136). Mitoma et al. (136) observed a minor degree of incorporation of hydroxyproline into protein in embryonic tissue, but this was overshadowed by the hydroxylation of the peptide-bound proline. 

Vitamin C is essential for the formation of collagen, the principal structural protein in skin, bone, tendons, and ligaments, being a cofactor in the hydroxylation of the amino acids proline to 4-hydroxyproline, and of lysine to 5-hydroxylysine. These hydroxyamino acids account for up to 25% of the collagen structure. Vitamin C is also associated with some other hydroxylation reactions, e.g. the hydroxylation of tyrosine to dopa (dihydroxyphenylalanine) in the pathway to catecholamines (see Box 15.3). Deficiency leads to scurvy, a condition characterized by muscular pain, skin lesions, fragile blood vessels, bleeding gums, and tooth loss. Vitamin C also has valuable antioxidant properties (see Box 9.2), and these are exploited commercially in the food industries. 

Ascorbic acid or vitamin C is found in fruits, especially citrus fruits, and in fresh vegetables. Man is one of the few mammals unable to manufacture vitamin C in the liver. It is essential for the formation of collagen as it is a cofactor for the conversion of proline and lysine residues to hydroxyproline and hydroxylysine. It is also a cofactor for carnitine synthesis, for the conversion of folic acid to folinic acid and for the hydroxylation of dopamine to form norepinephrine. Being a lactone with two hydroxyl groups which can be oxidized to two keto groups forming dehydroascorbic acid, ascorbic acid is also an anti-oxidant. By reducing ferric iron to the ferrous state in the stomach, ascorbic acid promotes iron absorption. 

Hydroxyproline and hydroxylysine Collagen contains hydroxy proline (hyp) and hydroxylysine (hyl), which are not present in most other proteins. These residues result from the hydroxylation of some of the proline and lysine residues after their incorporation into polypeptide chains (Figure 4.6). The hydroxylation is, thus, an example of posttranslational modification (see p. 440). Hydroxy proline is important in stabilizing the triple-helical structure of colla gen because it maximizes interchain hydrogen bond formation. 

Perret, S., Merle, C., Bernocco, S., Berland, P., Garrone, R., Hulmes, D.J., Theisen, M., and Ruggiero, F. (2001). Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation. J. Biol. Chem. 276, 43693-43698. 

Figure 3.4 Factors affecting foreign body reaction and potential points of intervention at the level of the myofibroblast (1) inhibit synthesis or release of TGF-P (2) block stimulation by TGF-P of its membrane receptors on the activated fibroblast (3) inhibit the Smad proteins, which transfer the TGF-P effect to the nucleus (4) inhibit transcription of procollagen mRNA (5) inhibit translation of the message to form procollagen (6) inhibit prolyl-4-hydroxylase, which creates hydroxyproline and facilitates helix formation (7) inhibit lysyl oxidase, which cross-links the collagen (8) enhance the function of MMPs, which degrade collagen, or inhibit TIMPs, which degrade MMPs.

It is conceivable that the reduced formation of tritiated water observed in scurvy could be due to a general reduction in protein synthesis, but this would not explain the marked differences between the specific activities of proline and hydroxyproline observed, nor would it explain observations in our laboratory that glycine and tyrosine are incorporated at rates similar to that of incorporation of proline into the collagen of granuloma obtained from scorbutic animals. 

The striking increases in the formation of tritiated water and tritiated hydroxyproline on in vitro addition of ascorbate are consistent with a function of this vitamin in hydroxylation—probably at step 3. The present results do not support a systemic ascorbic acid-mediated effect, the belief that ascorbic acid functions in the maintenance of collagen, or acts by stimulating maturation of the fibroblasts in the system under study here. The present data do not support the possibility that intermediates containing hydroxyproline accumulate in scurvy. The proposal that ascorbic acid is involved in the hydroxylation reaction itself is consistent with studies on the nonenzymatic hydroxylation of proline (4) and on enzymatic hydroxylation of other compounds (5, 20, 21, 44, 51). 

There is evidence that free hydroxyproline can be incorporated into collagen to some extent. Thus, Mitoma (26) found that free hydroxyproline was incorporated into chick embryo collagen at about 10% of the rate observed with proline. The scheme given above indicates a pathway by which there may be a relatively slow influx of hydroxyproline into the hydroxyproline intermediate. Similarly, the breakdown of this intermediate might be responsible for the formation of free hydroxyproline observed in some tissues and possibly also that found in urine. 

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