Paracrine signals from mesenchymal cell populations govern the expansion and differentiation of human hepatic stem cells to adult liver fates

Differentiation of embryonic or determined stem cell populations to adult liver fates under known conditions yields cells with some but not other adult-specific genes, aberrant regulation of one or more genes, and variation in the results from experiment to experiment. We tested the hypothesis that sets of signals produced by freshly isolated, lineage-dependent mesenchymal cell populations would yield greater efficiency and reproducibility in driving differentiation of human hepatic stem cells (hHpSCs) to adult liver fates. Subpopulations of liver-derived mesenchymal cells, purified by immunoselection technologies, included 1) angioblasts; 2) mature endothelia; 3) hepatic stellate cell precursors; 4) mature stellate cells (pericytes) and 5) myofibroblasts. Freshly immunoselected cells of each of these subpopulations were established in primary cultures under wholly defined (serum-free) conditions that we developed for short-term cultures and used them as feeders with hHpSCs. Feeders of angioblasts yielded self-replication; stellate cell precursors caused lineage restriction to hepatoblasts; mature endothelia produced differentiation to hepatocytes; and mature stellate cells and/or myofibroblasts resulted in differentiation to cholangiocytes. Paracrine signals, produced by the different feeders, were identified by biochemical, immunohistochemical, and qRT-PCR analyses and then those signals were used to replace the feeders in monolayer and 3-D cultures to elicit the desired biological responses from the hHpSCs. The defined paracrine signals proved able to yield reproducible responses from the hHpSCs and to permit differentiation to fully mature and functional parenchymal cells

Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin

Lysyl hydroxylase 3 (LH3) is a multifunctional protein with lysyl hydroxylase, galactosyltransferase and glucosyltransferase activities. The LH3 has been shown to modify the lysine residues both in collagens and also in some collagenous proteins. In this study we show for the first time that LH3 is essential for catalyzing formation of the glucosylgalactosylhydroxylysines of mannan-binding lectin (MBL), the first component of the lectin pathway of complement activation. Furthermore, loss of the terminal glucose units on the derivatized lysine residues in mouse embryonic fibroblasts lacking the LH3 protein leads to defective disulphide bonding and oligomerization of rat MBL-A, with a decrease in the proportion of the larger functional MBL oligomers. The oligomerization could be completely restored with the full length LH3 or the amino-terminal fragment of LH3 that possesses the glycosyltransferase activities. Our results confirm that LH3 is the only enzyme capable of glucosylating the galactosylhydroxylysine residues in proteins with a collagenous domain. In mice lacking the lysyl hydroxylase activity of LH3, but with untouched galactosyltransferase and glucosyltransferase activities, reduced circulating MBL-A levels were observed. Oligomerization was normal, however and residual lysyl hydroxylation was compensated in part by other lysyl hydroxylase isoenzymes. Our data suggest that LH3 is commonly involved in biosynthesis of collagenous proteins and the glucosylation of galactosylhydroxylysines residues by LH3 is crucial for the formation of the functional high-molecular weight MBL oligomers

Unusual Fragmentation Pathways in Collagen Glycopeptides

Collagens are the most abundant glycoproteins in the body. One characteristic of this protein family is that the amino acid sequence consists of repeats of three amino acids –(X—Y—Gly)(n). Within this motif, the Y residue is often 4-hydroxyproline (HyP) or 5-hydroxylysine (HyK). Glycosylation in collagen occurs at the 5-OH group in HyK in the form of two glycosides, galactosylhydroxylysine (Gal-HyK) and glucosyl galactosylhydroxylysine (GlcGal-HyK). In collision induced dissociation (CID), collagen tryptic glycopeptides exhibit unexpected gas-phase dissociation behavior compared to typical N- and O-linked glycopeptides, i.e. in addition to glycosidic bond cleavages, extensive cleavages of the amide bonds are observed. The Gal- or GlcGal- glycan modifications are largely retained on the fragment ions. These features enable unambiguous determination of the amino acid sequence of collagen glycopeptides and the location of the glycosylation site. This dissociation pattern was consistent for all analyzed collagen glycopeptides, regardless of their length or amino acid composition, collagen type or tissue. The two fragmentation pathways – amide bond and glycosidic bond cleavage – are highly competitive in collagen tryptic glycopeptides. The number of ionizing protons relative to the number of basic sites (i.e. Arg, Lys, HyK and N-terminus) is a major driving force of the fragmentation. We present here our experimental results and employ quantum mechanics calculations, to understand the factors enhancing the labile character of the amide bonds and the stability of hydroxylysine glycosides in gas phase dissociation of collagen glycopeptides