Cloning and characterization of a third human lysyl hydroxylase isoform

Abstract

Lysyl hydroxylase (EC 1.14.11.4), a homodimer, catalyzes the formation of hydroxylysine in collagens. Recently, an isoenzyme termed lysyl hydroxylase 2 has been cloned from human sources [M. Valtavaara, H. Papponen, A.-M. Pirttilä, K. Hiltunen, H. Helander and R. Myllylä (1997) J. Biol. Chem. 272, 6831-6834]. We report here on the cloning of a third human lysyl hydroxylase isoenzyme, termed lysyl hydroxylase 3. The cDNA clones encode a 738 amino acid polypeptide, including a signal peptide of 24 residues. The overall amino acid sequence identity between the processed human lysyl hydroxylase 3 and 1 polypeptides is 59%, and that between the processed lysyl hydroxylase 3 and 2 polypeptides is 57%, whereas the identity to the processed Caenorhabditis elegans polypeptide is only 45%. All four recently identified critical residues at the catalytic site, two histidines, one aspartate, and one arginine, are conserved in all these polypeptides. The mRNA for lysyl hydroxylase 3 was found to be expressed in a variety of tissues, but distinct differences appear to exist in the expression patterns of the three isoenzyme mRNAs. Recombinant lysyl hydroxylase 3 expressed in insect cells by means of a baculovirus vector was found to be more soluble than lysyl hydroxylase 1 expressed in the same cell type. No differences in catalytic properties were found between the recombinant lysyl hydroxylase 3 and 1 isoenzymes. Deficiency in lysyl hydroxylase 1 activity is known to cause the type VI variant of the Ehlers-Danlos syndrome, and it is therefore possible that deficiency in lysyl hydroxylase 3 activity may lead to some other variant of this syndrome or to some other heritable connective tissue disorder.

Figure 1

Alignment of the amino acid residues of the processed human lysyl hydroxylase 3 (Lh3), 1 (Lh1), and 2 (Lh2) polypeptides, and the C. elegans lysyl hydroxylase (C.e.Lh) polypeptide. Gaps (small dots) are introduced for the maximal alignment. Positions of cysteine residues (large dots) and potential N-glycosylation sites (solid lines) in any of the polypeptides are indicated above the sequence. Residues required for the binding of the Fe²⁺ atom and the C-5 carboxyl group of 2-oxoglutarate are indicated by asterisks. White letters on a black background show identity and black letters on a grey background show similarity. Similar amino acids: G, A, S; A, V; V, I, L, M; I, L, M, F, Y, W; L, K, H; D, E, Q, N; S, T, E, N.

Figure 2

Northern blot analysis of lysyl hydroxylase 3 mRNA in human tissues and cells. Each lane in A–C contains 2 μg of poly(A)⁺ RNA from the adult (A and B) or fetal (C) tissue indicated. Each lane in D contains 15 μg of total RNA from adult human Saos-2 osteosarcoma cells or skin fibroblasts. Blots were hybridized with a 354-bp PCR fragment from the 3′ end of the cDNA. Autoradiography time was 4 (A–C) or 7 (D) days.

Figure 3

Analysis of the expression of the human lysyl hydroxylase 3 polypeptide in insect cells by SDS/PAGE under reducing conditions. (A) NP-40 soluble proteins. (B) Glycerol buffer soluble proteins. (C) Proteins solubilized from the remaining pellets with 1% SDS. Lanes 1–5, samples from High Five cells infected with the virus coding for lysyl hydroxylase 3 and harvested 24, 40, 48, 64, and 72 hr after infection, respectively. Arrow indicates the location of the lysyl hydroxylase 3 polypeptides. Gels were stained with Coomassie brilliant blue.