Lysine post-translational modifications of collagen

Abstract

Type I collagen is the most abundant structural protein in vertebrates. It is a heterotrimeric molecule composed of two α1 chains and one α2 chain, forming a long uninterrupted triple helical structure with short non-triple helical telopeptides at both the N- and C-termini. During biosynthesis, collagen acquires a number of post-translational modifications, including lysine modifications, that are critical to the structure and biological functions of this protein. Lysine modifications of collagen are highly complicated sequential processes catalysed by several groups of enzymes leading to the final step of biosynthesis, covalent intermolecular cross-linking. In the cell, specific lysine residues are hydroxylated to form hydroxylysine. Then specific hydroxylysine residues located in the helical domain of the molecule are glycosylated by the addition of galactose or glucose-galactose. Outside the cell, lysine and hydroxylysine residues in the N- and C-telopeptides can be oxidatively deaminated to produce reactive aldehydes that undergo a series of non-enzymatic condensation reactions to form covalent intra- and inter-molecular cross-links. Owing to the recent advances in molecular and cellular biology, and analytical technologies, the biological significance and molecular mechanisms of these modifications have been gradually elucidated. This chapter provides an overview on these enzymatic lysine modifications and subsequent cross-linking.

Figure 1. Type I collagen biosynthesis and lysine modifications

The top part of the Figure (above the cell membrane) illustrates the intracellular events and the bottom part of the Figure (below the cell membrane) illustrates the extracellular events. During the synthesis of proα chains in the ER, specific peptidyl lysine residues are hydroxylated to form hydroxylysine (-OH-NH₂) and, subsequently, specific glycosylated hydroxylysine residues (O-linked glycosylation). For the latter, either single galactose (a red hexagon) or glucose-galactose (two red hexagons) is attached. After these and other modifications (e.g. hydroxylation of proline, asparagine-linked glycosylation shown as closed circles in the C-propeptide), two proα1 chains (solid line) and one proα2 chain (dotted line) associate with one another and fold into a triple helical molecule from the C- to the N-terminus to form a procollagen molecule, packaged and secreted into the extracellular space. Then both N- and C-propeptides are cleaved to release a collagen molecule. The collagen molecules are then spontaneously self-assembled into a fibril and stabilized by covalent intra- and inter-molecular covalent cross-linking. During fibrillogenesis, molecules are packed in parallel and are longitudinally staggered by an axial repeat distance, D period (~67 nm) creating two repeated regions, i.e. overlap and hole regions, in the fibril.

Figure 2. The structure of type I procollagen

Type I procollagen is composed of two proα1(I) chains (solid line) and one proα2(I) chain (dotted line) forming a unique triple helix. The N-propeptide contains a short triple helical domain and intrachain-disulfide bonds. Each chain of the globular C-propeptide is glycosylated by high-mannose asparagine-linked oligosaccharide (man)_n and this domain is stabilized by interchain-disulfide bonds (lines). The procollagen molecule is cleaved at specific sites at both N- and C-terminal regions (indicated by arrows) by ADAMTS and BMP1/Tolloid-like proteinases respectively. For the illustrations of hydroxylysine and its glycosylation, see Figure 1. Type I collagen is a ~300 nm long and ~1.5 nm thick molecule containing three domains: N-telopeptide (N-telo), uninterrupted triple helix and C-telopeptide (C-telo). Each α chain in the molecule is coiled into an extended left-handed helix and, then, three α chains are folded into a right-handed triple helix structure. Gal, galactose unit; Glc, glucose unit.

Figure 3. Lysine modifications in the helical domain of type I collagen

(A) Molecular distribution of lysine residues in the helical domain of α1 (solid line) and α2 (broken line) chains based on human type I collagen. There are 36 lysine residues in an α1 chain and 30 lysine residues in an α2 chain. Grey squares indicate the relative positions of lysine residues in each chain and black squares indicate the intermolecular cross-linking sites (α1, residues 87 and 930; α2, residues 87 and 933). (B) Enzyme-mediated sequential modifications of helical lysine residues: (1) lysine hydroxylation forming hydroxylysine; (2) galactosylation of hydroxylysine forming G-Hyl; and (3) glucosylation of G-Hyl forming GG-Hyl. For details of the enzymes involved in each reaction, please see the text.

Figure 4. Lysine modifications in the telopeptide domains of type I collagen

There are five lysine or hydroxylysine residues in the telopeptide domains of type I collagen, two in the C- (two α1–16^C) and three in the N- (two α1–9^N and one α2–5^N) telopeptide, indicated by open circles. These five lysine/hydroxylysine residues can be oxidatively deaminated by a copper (Cu²⁺)-dependent amine oxidase, LOX, to form the respective aldehydes, i.e. Lys^ald and Hyl^ald. Solid line, α1 chain; broken line, α2 chain. For details, please see the text.

Figure 5. LOX-mediated collagen cross-linking

The top panel (boxed) shows the cross-linking sites of type I collagen. Black lines within and between the molecules indicate examples of the intra- and inter-molecular cross-linkages. Numbers in parentheses indicate the residue numbers of the telopeptidyl aldehydes (open circles) and the helical lysine or hydroxylysine residues (closed circles) involved in cross-linking. Red hexagon, galactose or glucose attached to the helical hydroxylysine involved in cross-linking; telo, telopeptide. Solid line, α1 chain; broken line, α2 chain. The bottom panel summarizes initiation, maturation and various pathways of cross-linking. The boxed cross-link compounds are non-reducible cross-links. t, telopeptidyl; hel, helical; ald, aldehyde; d-, deoxyde-; H, dehydro.