Data comparing the kinetics of procollagen type I processing by bone morphogenetic protein 1 (BMP-1) with and without procollagen C-proteinase enhancer 1 (PCPE-1)

This article provides kinetic constants for C-terminal processing of procollagen type I by bone morphogenetic protein 1 (BMP-1; the major procollagen C-proteinase), a reaction stimulated by the connective tissue glycoprotein procollagen C-proteinase enhancer 1 (PCPE-1). Reported are Km, Vmax, Kcat and Kcat/Km (catalytic coefficient) values for BMP-1 alone, BMP-1 with intact PCPE-1, BMP-1 with the CUB (Complement C1r/C1s, Uegf, BMP-1) domains fragment of PCPE-1 as well as its NTR (netrin-like) domain.


a b s t r a c t
This article provides kinetic constants for C-terminal processing of procollagen type I by bone morphogenetic protein 1 (BMP-1; the major procollagen C-proteinase), a reaction stimulated by the connective tissue glycoprotein procollagen C-proteinase enhancer 1 (PCPE-1). Reported are K m , V max , K cat and K cat /K m (catalytic coefficient) values for BMP-1 alone, BMP-1 with intact PCPE-1, BMP-1 with the CUB (Complement C1r/C1s, Uegf, BMP-1) domains fragment of PCPE-1 as well as its NTR (netrin-like) domain.
& 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

How data was acquired
Measurements of the rate of release of the C-propeptide from procollagen type I by bone morphogenetic protein 1 (BMP-1) at different procollagen concentrations and calculation of kinetic constants using Lineweaver-Burk plots and in part also the Michaelis-Menten equation using Graphpad software. Data format Analyzed Experimental factors Chick embryo tendon procollagen type I was labeled biosynthetically with H 3 -tryptophan. The CUB (Complement C1r/C1s, Uegf, BMP-1) and netrin-like (NTR) domains fragments of PCPE-1 were generated by limited proteolysis with trypsin and isolated by heparin-Sepharose chromatography. Experimental features Proteolytic processing was quantified by measuring the amount of radioactivity in the procollagen C-propeptide released by BMP-1.

Data source location
Tel Aviv University, Tel Aviv, Israel Data accessibility Data is provided within this article

Value of data
Researchers can use the data in designing in vitro assays of procollagen C-proteinase activity, in particular range of procollagen, BMP-1 and PCPE-1 concentrations.
The data extends understanding of the mode of action of PCPE-1 and thus could suggest new research directions.
The data rejects an early contradictory report on how PCPE-1 affects the kinetics of procollagen type I processing by BMP-1, thereby resolving a controversy in the field, also important as a guideline for future research.

Data
The data include a Figure depicting the initial rates of procollagen I cleavage by BMP-1 in the absence and presence of intact PCPE-1, its CUB domains fragment and its NTR domain as a function of procollagen concentration (Fig. 1). Also included are a Figure displaying the corresponding Lineweaver-Burk plots (Fig. 2) and a Table presenting the resulting K m , V max , K cat values and the related catalytic coefficients (K cat /K m ) ( Table 1).

Proteins
3 H-tryptophan-labeled procollagen type I (specific activity 10.36 Â 10 6 cpm/mg) was prepared from chick embryo tendon fibroblasts [1]. The specific activity of the C-propeptide (4.7 Â 10 7 cpm/mg) was calculated assuming the molecular weight of the C-propeptide is 22% of that of intact procollagen. Recombinant human PCPE-1 was prepared as previously described [2]. The NTR and CUB1CUB2 domains of PCPE-1 were generated from it by limited proteolysis and isolated by chromatography on a heparin-Sepharose column [3]. Recombinant human BMP-1 was expressed in insect cells (baculovirus system) and purified as described [4].

Assay of procollagen C-proteinase activity and determination of kinetic constants
Procollagen C-proteinase activity of BMP-1 was determined at procollagen concentrations ranging from 22.2 to 133.2 nM (10-60 μg/ml) and a constant BMP-1 concentration of 0.14 nM estimated from relative band intensity after SDS-PAGE and silver staining. Due to the limited accuracy of such estimation the actual concentration of BMP-1 could be up to 50% higher or lower than the above value of 0.14 nM but this does not affect the ratios between kinetic constants with and without PCPE-1, the information we sought. Reaction conditions and measurements of the amount of radioactivity in the C-propeptide were as previously described [1,5]. PCPE-1 and its fragments (when added to the reaction solutions) were used at 15 μg/ml (0.3, 0.5 and 0.75 μM for intact PCPE-1, the CUB1CUB2 fragment and the NTR fragment, respectively; molar ratios relatively to the highest procollagen Table 1 Kinetic constants for procollagen type I processing by BMP-1 in the absence or presence of various PCPE-1 forms. None (BMP-1 alone) 3.9 71. Data are presented as mean 7 standard error n ¼ 6 for BMP-1 alone and 2 for each of the other conditions. The differences between the K m and V max values for BMP-1 alone and BMP-1 plus NTR are statistically insignificant (p¼0.11 and 0.51, respectively) and so are the differences between the K m values for BMP-1 plus PCPE-1 and BMP-1 plus its CUB domains fragment (p¼ 0.095). The difference between the corresponding V max values is statistically significant (p¼0.004). Differences between V max for BMP-1 alone and BMP-1 plus PCPE-1 or BMP-1 plus its CUB domains are statistically significant (p¼0.00097 and 0.0014, respectively). K cat values were calculated assuming the enzyme concentration was 0.14 nM. This value however was estimated based on the relative band intensity after SDS-PAGE and silver staining with an estimated error of up to 50%. Since all of the assays were performed using identical aliquots of BMP-1, the comparison between the respective K cat and K concentration were 2.25, 3.7 and 5.6:1, respectively). This ensured that the amount of each of the PCPE-1 forms added would exceed saturation required to achieve maximal activity. The enzyme input was selected based on preliminary assays, ensuring that the reaction rates remained linear at all procollagen concentrations both in the absence and presence of PCPE-1. Reactions were initiated by enzyme addition and were conducted in duplicates both with and without BMP-1 to determine (and subtract) the background values at each procollagen concentration. The amount of radioactive Cpropeptide released into the supernatants was calculated based on its specific activity. The kinetic constants K m and V max were derived from Lineweaver-Burk plots Fig. 2 and in the case of reactions in the presence of intact PCPE-1 and its CUB domains fragment, also based on the Michaelis-Menten equation achieved by fitting to the experimental data using Prism 5.0 (Graphpad software). This software did not allow calculations of kinetic constants for either BMP-1 alone or BMP-1 plus the NTR fragment, which required prohibitively high procollagen concentrations.

Statistical analysis
Data are presented as means7SD (standard deviation). Statistical significance was determined using two tail paired t-tests. Differences between groups were considered significant for p values o0.05.