Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort

Abstract

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340 nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6–3.9 and 7–20 nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9 months when stored at -80 °C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Introduction

N-glycosylation is a site-specific, multi-step enzymatic process that occurs in the cytoplasm, endoplasmic reticulum, and golgi apparatus [1]. The biosynthesis of all N-glycans occurs coherently and is essential in varied biological processes like protein folding, cell–cell interaction etc. Defects in glycosylation are implicated in several genetic and chronic diseases and complete absence of N-glycans is fatal [1]. Congenital disorders of glycosylation (CDG) are a group of over 100 different clinically and genetically heterogeneous glycosylation defects and ~ 50% of them are related to N-glycosylation disorders [2]. These glycosylation defects are distinct disorders and are not influenced by non-enzymatic glycation as seen in hyperglycemia. They are detected by transferrin isoform analysis wherein an increased disialo and/or asialo transferrin suggests type I defects while increase in tri, di, mono and/or asialo transferrin is indicative of type II N-glycosylation disorders [3].

Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI) are primary cytosolic enzymes involved in N-glycosylation. PMM catalyzes the reversible conversion of mannose-6-phosphate to mannose-1-phosphate and MPI converts fructose-6-phosphate to mannose-6-phopshate. Deficiency of these enzymes causes PMM2-CDG and MPI-CDG respectively. PMM2-CDG is the most common CDG and accounts for ~ 80% of cases while MPI-CDG is a treatable disorder and can be managed with mannose therapy [4]. These defects are identified by a type I transferrin isoform pattern and diagnosed by molecular testing of biallelic pathogenic variants in PMM2 or MPI gene. However, if these variants are of uncertain significance (vus), enzyme activity of PMM or MPI from leukocytes or fibroblasts would help in confirmation of diagnosis [4].

These enzyme assays are tedious and require expertise, hence, their availability is restricted to a few referral laboratories worldwide. In India, transferrin isoform analysis is available for screening [5] followed by molecular facilities for confirmatory diagnosis of CDG. The next generation sequencing (NGS) approach may result in identifying variants in several genes which may also include novel variants. In such cases respective enzyme assays may assist in confirmation of the inherited defect. Thus, we aimed to optimize PMM and MPI enzyme assays from leukocytes which would help to confirm the diagnosis of PMM2-CDG and MPI-CDG.

Materials and Methods

The study was carried out in the Biochemistry section of our hospital and was approved by the Institutional Ethics Committee.

Subjects

Healthy, non-alcoholic controls (n = 50, 31 females/19 males, mean age 36.3 y) and 20 pediatric patients (10 females/10 males, mean age 38 months) were recruited for the study. Since chronic alcoholics exhibit a transferrin isoform pattern similar to that observed in CDG type I defects [6], these subjects were excluded from the study group. All the participants were also screened for N-glycosylation disorders and subjects with a normal transferrin isoform pattern were included in the study. Heparinized blood samples were collected for standardization/validation of enzyme assays and establishing reference range amongst Indians. A written consent was obtained from all the healthy volunteers and from parents/guardian of pediatric patients prior to participation.

Chemicals

Ammonium chloride, Sodium bicarbonate, EDTA, Magnesium chloride and Sodium chloride were obtained from Merck (Kenilworth, NJ, US). Pierce ™ Bicinchoninic Acid (BCA) Kit for protein estimation from Thermo Fisher Scientific (Waltham, MA, US). Mannose-6-Phosphate, Mannose-1-Phosphate, Glucose 1,6 Bisphosphate, Phosphoglucoisomerase, Phosphomannose isomerase, Glucose-6-Phosphate dehydrogenase, NADP and HEPES buffer were purchased from Sigma Aldrich (St. Louis, MO, US). All chemicals were of analytical grade and were prepared in Milli-Q water from Elix®Milli Pore system.

Methods

Preparation of Cell Extracts

Blood sample (3 ml) was collected from participants and leukocytes were isolated within 24 h of sample collection according to the method of Roos and Loos with minor alterations [7]. Samples were centrifuged at 3000 rpm for 10 min and the buffy layer was collected for leukocyte isolation. RBC lysis buffer (155 mM ammonium chloride, 10 mM sodium bicarbonate, 0.1 mM EDTA) and isotonic saline (0.9% sodium chloride) were used to lyse and remove erythrocytes by cyclic centrifugation at 2500 rpm for 10 min at 4 °C. The washed leukocyte pellets were stored immediately at -80 °C until analysis.

The leukocyte pellets were lysed prior to enzyme assays. For this, pellets were thawed on ice and homogenized with 100 µl of chilled homogenization buffer (50 mM HEPES pH 7.1, 5 mM MgCl₂) using a pre-cooled teflon pestle. Protein concentration was estimated spectrophotometrically at 562 nm using commercially available BCA kit with bovine serum albumin standard and was adjusted to 3 mg/ml with homogenization buffer for enzyme analysis.

Enzyme Assays

PMM and MPI enzymes were measured in leukocyte as described by Van Schaftingen E and Jaeken J [8]. with slight modifications. For PMM assay, 80 µl of cell-extract was added to a reaction mixture containing 50 mM HEPES pH 7.1, 5 mM MgCl₂, 100 µM Mannose-1-Phosphate and 100 µM Glucose-1,6-bisphosphate. The mixture was incubated at 37 °C for 90 min after which the reaction was stopped by heating at 80 °C for 5 min and snap cooling on ice. The mixture was then centrifuged at 3000 rpm for 10 min and supernatant was collected. To this supernatant, 10 µg/ml Phosphoglucoisomerase, 3.5 µg/ml Phosphomannose isomerase, 10 µg/ml glucose 6 phosphate dehydrogenase and 1 mM NADP were added for enzyme activity. For MPI assay, a mixture containing 50 mM HEPES pH 7.1, 5 mM MgCl₂, 0.5 mM Mannose-6-Phosphate, 10 µg/ml Phosphoglucoisomerase, 2 µg/ml glucose-6-phosphate dehydrogenase, 0.25 mM NADP and 20 µl of cell extract was used.

The enzyme activities were estimated spectrophotometrically by monitoring the conversion of NADP to NADPH at 340 nm. Absorbance was measured on a Beckman Coulter (DU® 800) spectrophotometer in a continuous auto-reading mode and temperature was controlled at 37ºC for optimal enzymatic reaction. Readings were recorded at 1 min intervals for a total reaction time of ten minutes with first time (zero) measurement being read immediately after adding NADP and last reading at the end of 10^th minute. 1U of enzyme activity corresponds to formation of 1 µmol of NADPH.

Quality Control

Pooled leukocyte extracts were used for inter-assay quality control. Background interference from reagents and lysate was assessed by including a reagent blank (without substrate) and a heat inactivated cell lysate in every batch. The sample integrity was ensured by performing both the enzyme assays from the same pellet as each enzyme worked as a reference enzyme for the other.

Sample Viability and Enzyme Stability

Sample viability was assessed by collecting 2 blood sample tubes from the same individuals and isolating the leukocyte a) within 24 h and b) after allowing it to stand at room-temperature for 3 days. The viability studies were done from samples collected from two adult participants. Enzyme stability in the leukocyte pellet was assessed by estimating the enzyme activities from pooled QC leukocytes at 12 weekly intervals up to 9 months.

Statistical Analysis

Data was recorded in MS Excel. All the statistical calculations were performed using MedCalc version 18.11.6. Statistically significant change was considered to occur when p is less than 0.05.

Results

Reference Range

The enzyme activities were estimated for 50 healthy controls and 20 pediatric patients. The PMM activity was in the range of 1.6–3.9 nmol/min/mg protein and MPI activity ranged from 7 to 20 nmol/min/mg protein. Enzyme range for the mean enzyme values were similar in males (PMM: 2.5 ± 0.5 nmol/min/mg protein; MPI: 11.7 ± 3.9 nmol/min/mg protein) and females (PMM: 2.7 ± 0.5 nmol/min/mg protein; MPI: 11.6 ± 3.4 nmol/min/mg protein) with no statistically significant difference (PMM: p = 0.121; MPI: p = 0.659). Enzyme activities for adults and pediatric subjects were overlapping and did not exhibit any variation (Table 1).

Table 1.

Reference range of PMM and MPI enzymes for paediatric and adult participants

Study controls	PMM (nmol/min/mg protein)	MPI (nmol/min/mg protein)
Paediatric group (n = 20)	2.2–3.0	7.8–17.5
Adult group (n = 50)	1.6–3.9	7.0–20.0
P value	0.344	0.562

Quality Control

The between run quality control checks using pooled leukocytes showed %CV for PMM and MPI to be 19.9% and 19.8% respectively.

Sample Viability and Enzyme Stability

The enzyme activities in the samples wherein leukocytes were isolated within 24 h of withdrawal were within the normal ranges for PMM and MPI while that from samples allowed to stand at room-temperature showed a significant loss of activity for both the enzymes due to hemoglobin interference. This occurred due to hemolysis of the sample on standing. The isolated leukocyte pellets when stored at -80 °C were stable for up to 9 months wherein the %CV for initial activities and after every 12 weeks up to 9 months was 17.5% and 16% for PMM and MPI respectively.

Discussion

Unavailability of screening and diagnostic facilities has limited detection of CDG in India. An earlier study from our center on transferrin isoform analysis identified a Type I pattern in 2 of the 50 patients clinically suspected of having CDG [5]. Availability of isoform analysis and next generation sequencing in India may help to identify CDG however, enzyme assays are needed to further subtype CDG and also phenotypically confirm the variant in respective genes.

Evaluation of enzyme levels from leukocytes, lymphoblasts and/or fibroblasts in healthy individuals and CDG patients have been primarily performed in European population [8–11]. Amongst the healthy controls, fibroblast PMM & MPI enzyme activity (n = 5) of 1.32 ± 0.37 mU/mg protein and 14.6 ± 4.1 mU/mg protein respectively has been reported by Van Schaftingen E and Jaeken J [8]. Subsequently, Barnier A et al., [9] documented leukocyte PMM enzyme activity in 376 healthy individuals of more than 2 years of age in the range of 4.8 ± 1.4 mU/mg protein. Jaeken J et al., have measured enzyme activity from leukocytes, fibroblasts and lymphocytes [10]. The enzyme activities in these cells were well differentiated i.e. 1.8–3.2 mU/mg protein from leukocytes, 3–5.7 mU/mg protein from fibroblasts and 7.3–11 mU/mg protein from lymphoblasts. PMM activity of 1.24 ± 0.91 nmol/min/mg protein from 58 controls has also been noted by Hansikova H et al., [11]. Similar enzyme activity has been described in Malaysians by Thong M et al., [12] wherein leukocyte PMM and MPI activity of 1.6–6.2 nmol/min/mg protein and 12–25 nmol/min/mg protein respectively were obtained. Our findings of leukocyte PMM and MPI activity in the range of 1.6–3.9 nmol/min/mg protein and 7–20 nmol/min/mg protein respectively show an overlap with the earlier reported ranges in different populations.

Loss of enzyme activity when leukocyte isolation is done from hemolysed samples has been observed by Barnier et al., [9]. Hemoglobin interference during measurement of NADPH has been reported by da Fonseca-Wollheim F.[13]. We have also noted a significant loss in enzyme activity due to hemoglobin interference thus suggesting that the leukocyte isolation should be done at the earliest from non-hemolysed samples. A significant reduction in PMM and MPI enzyme assays in CDG patients has been well reported [8–12]. The decrease in PMM activity in patients is remarkable in leukocytes with a residual activity of 10% of the controls in majority of the patients however a high residual activity up to 50% has also been observed in PMM2-CDG patients [11]. Thus, PMM and MPI assays aid in CDG diagnosis.

Our study is a preliminary report from India. We have studied leukocyte PMM and MPI activity in healthy individuals. The proposed method can be used for differential diagnosis of PMM2-CDG and MPI-CDG and detect the residual activity amongst Indian patients.