COLLAGEN SUBSTRATE SPECIFICITY OF PROLYL 3-HYDROXLASES

Alex W Farnand1,2, Mary Ann E Weis2, Lammy S Kim2, Russell J Fernandes2, David R Eyre2 1.  School of Medicine, University of Washington, Seattle, WA, USA.;

2. Orthopaedic Research Laboratories, Department of Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA.

BACKGROUND •  By weight, collagens are the most abundant protein in multi-

cellular animals. The unique supramolecular structure of collagen involves a number of post-translational modifications that are known to be catalyzed by specific enzymes. "

•  4-Hydroxyproline (4Hyp), a post-translational modification of the collagen strand, is known to stabilize the collagen triple helix. The precise function of 3Hyp, a much more rare hydroxyproline, is largely unknown, despite its discovery over fifty years ago. Ogle et al. J Biol Chem 237 (1962), pp. 3667-3673."

"•  Mutations in LEPRE1, a gene that encodes prolyl 3-

hydroxylase 1 (P3H1)—a protein responsible for 3-hydroxyproline (3Hyp) formation—was recently found to cause a recessive form of severe osteogenesis imperfecta. Cabral et al. Nat Genet, 39 (2007), pp. 359–365."

•  P3H1 is known to form a trimolecular complex with CRTAP (cartilage-associated protein) and cyclophilin B in the endoplasmic reticulum and is necessary for 3Hyp formation at the P986 prolyl residue in collagen. Vranka et al. J Biol Chem, 279 (2004), pp. 23615-23621."

•  3Hyp residues have recently been implicated as having a fundamental role in supramolecular collagen assembly by forming hydrogen bonds between adjacent collagen triple helices. Weis et al. J Biol Chem, 285 (2009), pp. 2580-2590."

The respective P3H enzymes—P3H1, P3H2 and P3H3—preferentially target specific prolyl residues in the collagen

fibril for the formation of 3Hyp residues."

1. RT-PCR analysis shows RCS-LTC cells exhibit full expression of all three P3H enzyme genes; other tissues show reduced or lack of expression in one or more P3H genes.

The high relative abundance of P3H2 mRNA expression coupled with high occupancy of Pro944

in the RCS-LTC matrix implicates P3H2 in the formation of 3Hyp at Pro944 supporting evolved substrate preferences for each of the three P3H

enzymes.

A B C

Funded under NCRR Grant TL1 RR 025016 and NIAMS Grant AR 036794

• The rat chondrosarcoma cell line, RCS-LTC, lays down an abundant matrix of collagen but fails to process this fine filamentous network beyond the stage of polymerized N-procollagen molecules and thus offers a unique way to study the effects of P3H enzyme activity.

• Using RT-PCR, the relative expression profiles of the P3H

enzyme genes and the genes of PPIB and CRTAP—other proteins related to the functionality of the P3Hs—were compared across RCS-LTC cells, normal-adult rat cartilage, the human osteosarcoma cell line, Saos-2 and in human fetal cartilage.

• Expression of the three P3H enzyme genes were assayed in RCS-LTC cells, and in normal-adult rat cartilage, via qPCR.

• Collagen produced by the RCS-LTC cells was analyzed for

3Hyp formation at previously identified prolyl residues by mass-spectometry.

HYPOTHESIS

APPROACH

RESULTS

SUMMARY CONCLUSION

!"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,&A. RCS-LTC Cells

!"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,&

B. Adult Normal Rat Cartilage

!"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,&C. Saos-2 Cells D. Human Embryonic Cartilage

!"#$%& ##'(& )*+"#& #,%-& #,%.& #,%,&

RCS-LTC cells produce a full complement of the genes assayed for; when compared to other cells and tissues, RCS-LTC cells also produce an extreme abundance of P3H2 (A). Adult Normal Rat Cartilage, similar to the RCS-LTC cells, produces all the genes assayed for; however, expression of P3H2 is semi-quantitatively less than the expression seen for P3H1 and P3H2 (B). Saos-2 cells completely lack expression of P3H2 (C). Human embryonic cartilage only produces P3H1 to a slight degree and fully lacks expression of P3H3 (D). "

2. RCS-LTC cells express markedly increased levels of P3H2 and P3H3 when compared to normal adult rat cartilage

3. RCS-LTC collagen showed near complete 3Hyp formation at Pro944.

• RT-PCR analysis of RCS-LTC cell total RNA showed that the cells express a full complement of the P3H enzyme genes, with

particularly robust expression of P3H2, a phenomenon unmatched by the other tissues investigated."• qPCR analysis of P3H enzyme gene expression in RCS-LTC cells and normal adult rat cartilage showed that the RCS-LTC

cells express all three enzymes to a greater extent than does the normal cartilage. Expression of P3H2, in particular, was significantly increased in the RCS-LTC cells."

• Mass-spectometric analysis of the RCS-LTC collagen showed near complete 3Hyp formation at Pro944, a secondary site previously found occupied in vitreous type II collagen, but not in cartilage"

Quantitative comparison of P3H mRNA expression between the RCS-LTC cells and rat cartilage yielded similar expression of P3H1, a ~2-fold increase in P3H2 and a ~0.5-fold increase in P3H3 expression in the RCS-LTC cells."

A1A4 A2A3

N C

A1A4 A2A3N C

Procollagen molecule

Fibril

P944

3Hyp sites

Rel

ativ

e Ab

unda

nce 100

020406080

RCS 1(II) P944 MS

400 600 800 1000 1200 1400 1600

m/z

1369.12+

1376.62+

y6

3Hyp

3Hyp

GFTGLQGLPGPPGPSGDQGTSGPAGPSGPR** #

20001800

Rel

ativ

e Ab

unda

nce 100

020406080

MSMS 13762+ ion

400 600 800 1000 1200 1400 1600 20001800

b8

y22

y20y17y10

y222+

y282+y13

1978.9

1807.8570.1

774.4882.4

989.7

1168.71274.7 1524.4

GFTGLQGLPGPPGPSGDQGTSGPAGPSGPR** #

y28 y6y10y13y17y20y22

b8

Pro

m/z

Tandem mass-spectrometric analysis of RCS-LTC cell collagen matrix (Figure A). The relative abundance of the ions shown provides an index of the degree of hydroxylation at Pro944 (top panel, fig. A). As shown, Pro944 is nearly 100% occupied by 3Hyp, which is most similar to vitreous type II collagen (87% occupied). The bottom panel of fig. A shows the MS/MS spectral analysis of the prolyl and suspected 3-hydroxyprolyl versions of the peptide, from which the y ion ladder establishes the position of the added 16 Da on Pro944. Figure B illustrates the potential 3Hyp sites on a procollagen molecule (top) and how axial relationships required for tri-functional intermolecular cross-links form in collagen to produce the collagen fibril (bottom)."

P3H1 P3H2 P3H30.0

0.5

1.02.0

2.5

RCS-LTC cellsRat Cartilage

***

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Rel

ativ

e E

xpre

ssio

n (!!

CT)

A B